Research ArticleN-Terminal Region of GbIspH1 Ginkgo bilobaIspH Type 1 May Be Involved in the pH-DependentRegulation of Enzyme Activity

Bok-Kyu Shin1 Joong-Hoon Ahn2 and Jaehong Han1

1Metalloenzyme Research Group and Department of Biotechnology Chung-Ang University Anseong 456-756 Republic of Korea2Department of Bioscience and Biotechnology BioMolecular Informatics Center Konkuk University Seoul 143-701 Republic of Korea

Correspondence should be addressed to Jaehong Han jaehonghcauackr

Received 24 January 2015 Accepted 28 February 2015

Academic Editor Qi Zhang

Copyright copy 2015 Bok-Kyu Shin et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

GbIspH1 IspH type 1 in Ginkgo biloba chloroplast is the FeS enzyme catalyzing the reductive dehydroxylation of HMBPP toisopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) at the final step of methylerythritol phosphate pathwayin chloroplast Compared to the bacterial IspH plant IspH including GbIspH1 has an additional polypeptide chain at the N-terminusHere biochemical function of theN-terminal region ofGbIspH1was investigatedwith theN-terminal truncatedGbIspH1(GbIspH1-truncated) Both wild type GbIspH1 (GbIspH1-full) and GbIspH1-truncated were catalytically active and produced IPPandDMAPP in a ratio of 15 1 Kinetic parameters of119870

119872(173plusmn 19 and 149plusmn 23120583M) and 119896cat (369plusmn 10 and 347plusmn 12minminus1) at pH80

were obtained for GbIspH1-full and GbIspH1-truncated respectively Interestingly GbIspH1-full and GbIspH1-truncated showedsignificantly different pH-dependent activities and the maximum enzyme activities were obtained at pH 80 and 75 respectivelyHowever catalytic activation energies (119864

119886) of GbIspH1-full and GbIspH1-truncated were almost the same with 365 plusmn 16 and 350 plusmn

19 kJmol respectively It was suggested that the N-terminal region of GbIspH1 is involved in the pH-dependent regulation ofenzyme activity during photosynthesis

1 Introduction

Since the discovery of mevalonate- (MVA-) independentmethylerythritol phosphate (MEP) pathway [1] significantattentions have been drawn to the new isoprenoids biosyn-thetic pathway due to the potential applications in medicine[2] Whereas MVA pathway is exclusively found in animalsAchaea and fungi MEP pathways are ubiquitous in mostbacteria protozoa and cyanobacteria [3] Interestingly plantis known to utilize both pathways as MVA and MEP path-ways are found in cytosol and plastid respectively In MVApathway mevalonate produced from acetyl-CoA is convertedto isopentenyl diphosphate (IPP) by three ATP-dependentenzymes However MEP pathway begins with the glycolysismetabolites pyruvate and glyceraldehyde-3-phosphate andseven unique enzymes comprise the pathway to produce IPPand dimethylallyl diphosphate (DMAPP) (see Figure 1)

In MEP pathway 4-hydroxy-3-methylbut-2-enyl diphos-phate (HMBPP) reductase (HDR IspH or LytB EC 11712)catalyzes the final step and the reductive dehydroxylationof HMBPP produces IPP and DMAPP [4 5] A 3 1 site-differentiated Fe

4S4cluster was identified at the active site

of bacterial IspH by Mossbauer spectroscopy [6] and theprotein X-ray crystallographic structures [7ndash9] provided thestructural feature of the substrate binding site At presenttwo different reaction mechanisms were proposed for IspHBothmechanisms involve the same reaction steps of substratebinding one electron reduction of the FeS cluster andthe electron transfer from the reduced [Fe

4S4]2+ cluster to

HMBPP However Birch reduction-like mechanism requiresformation of the allylic radical or anionic intermediate [1011] whereas the organometallic mechanism proposes the 1205782-alkenyl metallacycle intermediate formed between the FeScluster and HMBPP [12]

Hindawi Publishing CorporationBioinorganic Chemistry and ApplicationsVolume 2015 Article ID 241479 8 pageshttpdxdoiorg1011552015241479

2 Bioinorganic Chemistry and Applications

OH

OPP OPPOPP

HMBPP

IPPIDS DMAPP

2eminus2H+

+H2O

Figure 1

Most mechanistic studies were carried out with thebacterial IspH [13] but no biochemical reports are availablefor plant IspH Although plant IspH appears to followthe same reaction mechanism as bacterial IspH its aminoacid sequences are distinct from the bacterial IspH (seeFigure S1 in Supplementary Material available online athttpdxdoiorg1011552015241479) Namely plant IspHproteins have extra 100ndash130 amino acid residues at the N-terminal region To study the reaction mechanism of plantIspH GbIspH1 (GbIspH1-full) IspH type 1 from Ginkgobiloba was prepared Besides biochemical functions of theN-terminal region of the plant IspH were also investi-gated with the N-terminal truncated GbIspH1 (GbIspH1-truncated)

2 Methods

21 General Information Methyl viologen (MV) sodi-um dithionite (DT) trizma base N-cyclohexyl-2-aminoet-hanesulfonic acid (CHES) 4-(2-hydroxyethyl)-1-piperazi-neethanesulfonic acid (HEPES) 2-(N-morpholino)eth-anesulfonic acid (MES) and disodium salt of 551015840-bis-2-furansulfonic acid (ferene) were purchased fromAldrich For the anoxic environment (O

2lt 1 ppm) Schlenk

line and anaerobic chamber (MOTech) equipped withPd catalyst were employed Enzyme substrate HMBPPwas prepared according to the published method [14] andcharacterized by NMR spectroscope CD spectra of proteinsin the same buffer solution (571 120583M) were measured at therange of 190 nm to 260 nm with Chirascan plus (AppliedPhotophysics)

22 Expression and Purification of GbIspH1 GbIspH1 withHis-tag (GbIspH1-full) was obtained from recombinantEscherichia coli BL21 (DE3) containing pQE-31 vectorwhich was kindly provided by Professor Soo-Un Kim atSeoul National University Korea The N-terminal polypep-tide (Met1-Arg61) truncated GbIspH1 was subcloned intoBamHINotI sites of pETDuet (Novagen) His6-tag waslocated at the N-terminal of both proteins and GbIspH1-fulland GbIspH1-truncated were purified using His-tag affinitycolumn chromatography The amino acid sequences of twoproteins are available at Supplementary Material The cellwas grown in LB media containing 50120583gmL ampicillin10mM FeCl

3 and 10mM (NH

4)2SO4until the OD

600

reached 06 at 37∘C Cells were then incubated with 10mM

isopropyl-1-thio-120573-D-galactopyranoside (IPTG) at 30∘C for13 hours to express the proteins Cells were harvested bycentrifugation (3000 g 15min) at 4∘C and stored at minus70∘Cbefore use Frozen cells (typically 10 g) were thawed atroom temperature under nitrogen atmosphere and dilutedwith ten times volume of anaerobic lysis buffer (50mMphosphate pH 80 and 150mMNaCl) on ice Cell lysate wasobtained from sonication (10 cycles of 20 sec burst with 1-minute break) under argon atmosphereThe supernatant wascollected after centrifugation at 11000 g for 20minutes at 4∘CThe supernatant was loaded on a Ni-NTA affinity columnchromatography (4mL resin) in anaerobic chamber Theresin was preequilibrated with the phosphate buffer (50mMphosphate pH 80 and 150mM NaCl) Column was washedwith phosphate buffer containing 20mM imidazole and theneluted with phosphate buffer containing 200mM imidazoleFractions having a brown colorwere pooled and thendesaltedby Amicon ultra centrifugal filter (30 kDa) at 4000 g and thebuffer exchanged to the TrisHCl buffer (50mM TrisHClpH 80 and 100mM NaCl) UV-Vis spectra of proteinsare obtained from Scinco S-3150 UV-Vis spectrophotometerProtein concentrationwas determined by Bradford assay [15]The iron contents of the purified GbIspH1 proteins weredetermined by the absorption at 532 nm of Fe2+-ferene (dis-odium salt of 551015840-[3-(2-pyridyl)-124-triazine-56-diyl]bis-2-furansulfonic acid) complex [16]

23 Enzyme Kinetics of GbIspH1 Enzyme kinetics study ofGbIspH1-full and GbIspH1-truncated was carried out understrict anaerobic conditions To the reaction mixture (20mMMV 04mM DT 50mM HEPES and pH 80) containingHMBPP 200 nM of GbIspH1 was added to initiate thereaction Total volume of enzyme reaction mixture was450 120583L and the concentration of HMBPP was varied between0 and 250 120583M for substrate-dependent kinetics Reductionof HMBPP was monitored by the disappearance of reducedMV (120576

732= 3150Mminus1 cmminus1) at 732 nm by UV-Vis spectropho-

tometer Sigma plot software was used to obtain kineticsparameters from the initial reaction rates

For pH-dependent kinetics study GbIspH1 (200 nM) wasadded to the three-buffer reaction mixture (250 120583MHMBPP20mMMV 04mMDT 50mM CHES 50mMHEPES and50mM MES) after adjusting pH Temperature-dependentkinetics carried out with 250 120583M of HMBPP at 0ndash50∘CGbIspH1 (200 nM) was added to the reaction mixture to

Bioinorganic Chemistry and Applications 3

initiate the reaction while the required reaction temperaturewas maintained

24 Reaction Product Identification To isolate reaction prod-ucts enzyme reaction was carried out with GbIspH1 (1120583M)HMBPP (53mM)MV (50mM) andDT (75mM) in 300 120583Lof 50mM HEPES buffer (pH 80) at room temperature for3 h under anaerobic atmosphere After reaction ceriumoxide(075 g) in 300 120583L 02N ammonia solution was added to theenzyme reaction mixture [17] The solution was reacted for1 h at 40∘C with mild stirring and the dephosphorylatedproducts were extracted with ethyl acetate (200120583L times 3) ForGCMS analysis Agilent 7890A (GC) equipped with DB-5MS (19091S-433) column (30m 025mm and 025 120583m) wasconnected with Agilent 5975C (MSD) detector The sample(1 120583L) was injected to the injector (250∘C) in splitless modeTemperature of oven was kept at 40∘C for 5min beforeincreasing to 450∘C (10∘Cmin) Mass spectra were obtainedwith positive ionization mode by electron ionization (70 eV)

25 Sequence Analysis and Protein Structure PredictionAmino acid sequences of IspH proteins were aligned usingthe MultAlin program (httpmultalintoulouseinrafrmultalinmultalinhtml) Molecular weight and molarextinction coefficient of GbIspH1-full and GbIspH1-trun-cated were calculated using Protein Calculator v33 program(httpwwwscrippsedusimcdputnamprotcalchtml) Predic-tion of 3-dimensional protein structures of GbIspH1-full wasachieved by I-TASSER (httpzhanglabccmbmedumicheduI-TASSER) server

3 Results and Discussion

FeS proteins participate in many important biologicalprocesses such as photosynthesis and energy metabolismBesides the electron transfer FeS enzymes catalyze manyunique reactions of nitrogen fixation hydrogen productionCO oxidation and more Even simple cuboidal [4Fe

4S]

cluster acts as a cofactor in the radical SAMreaction and IspHreduction

Whereas extensive biochemical characterization of bac-terial IspH has been reported no enzyme kinetics on plantIspH are available Here we first reported catalytic propertiesof GbIspH1 Kim et al [18] suggested that the extra N-terminal peptide region contained the 10ndash40 residue-longtransit peptides whichmay facilitate the translocation of IspHinto chloroplast However biochemical function of the otherN-terminal polypeptide region between the transit peptideand catalytic domain is not clear yetThepurpose of this studywas to study the difference between plant IspH and bacterialIspH proteins and to investigate the possible function of theextra N-terminal polypeptide region of GbIspH1 The N-terminal truncated GbIspH1 used in this study was preparedby removing 61 amino acids at the N-terminus of GbIspH1

31 Purification and Characterization of GbIspH1 GbIspH1proteins of GbIspH1-full and GbIspH1-truncated were puri-fied by Ni-NTA affinity column chromatography and

the purity of proteins was confirmed by SDS-PAGE (FigureS2a) The apparent molecular weights of GbIspH1-full andGbIspH1-truncated estimated from the band positions were566 and 493 kDa which were close to the theoretical valuesof 560 and 497 kDa respectively UV-Vis spectra of thepurified GbIspH1-full and GbIspH1-truncated were obtainedunder anaerobic conditions and the absorptions at 410 nmA410

characteristic to the Fe4S4cluster were observed for

both (Figure S2b)TheA410

A280

ratio of the [Fe4S4]2+ cluster-

containing protein measured by UV-Vis spectrophotometerhas been used to evaluate the percentage of holoprotein in thepurified metalloproteinThe A

410A280

ratios of GbIspH1-fulland GbIspH1-truncated were measured to be 018 and 017respectively and both proteins did not require reconstitutionof the Fe

4S4cluster Iron contents of GbIspH1-full and

GbIspH1-truncated were also determined by colorimetricmethod using ferene and 31 plusmn 02 and 35 plusmn 03 Feproteinwere determined for GbIspH1-full and GbIspH1-truncatedrespectively The incorporation of the Fe

4S4cluster in the

purified GbIspH1 proteins was finally confirmed by EPRspectroscopy The methyl viologen- (MV-) reduced WFTshowed rhombic signals at 20K characteristic to the reduced[Fe4S4]+ cluster (Figure S3) CD spectra obtained from

GbIspH1-full and GbIspH1-truncated were very similar andindicated that both proteins were folded similarly (FigureS2c)TheCD spectra were also similar to those reported fromAaIspH [19] suggesting that IspH proteins from plant andbacteria are similar in their secondary structure composi-tions

The purification of GbIspH1-full and GbIspH1-truncatedproteins was carried out under oxygen-free conditions andthe identification of the FeS cluster required a few differentbiophysical characterizationsThe isolated proteins exhibitedpale brown color and the existence of the [Fe

4S4]2+ cluster

was evident from the electronic spectra With protein quan-titation and Fe analysis incorporation of the [Fe

4S4] cluster

in GbIspH1-full and GbIspH1-truncated was confirmed EPRspectroscopic study of the as-isolated and dithionite- (DT-) reduced proteins supported that the [Fe

4S4] cluster in

GbIspH1 shuttled +2 and +1 redox state

32 Enzyme Kinetics of GbIspH1 To measure the enzymeactivity of the purified IspH electrons from the elec-tron donor are required because IspH catalyzes the reduc-tive dehydroxylation of HMBPP A few in vitro elec-tron donors including biological NADPHflavodoxin reduc-taseflavodoxin and abiological DTMV reducing systemshave been utilized for the steady-state kinetics of IspH [11]In this study DT-reduced MV was used as an electrondonor and substrate-dependent reaction rates of GbIspH1-full and GbIspH1-truncated were measured in the presenceof HMBPP by spectrophotometry In detail one-electronreduced MV exhibits two strong electronic absorptionsat 732 nm (120576

732= 3150Mminus1 cmminus1) and 604 nm (120576

604=

18750Mminus1 cmminus1) and two molecules of reduced MV areconsumed for the product formation by IspH The elec-tron transfer and HMBPP reduction are tightly coupled inthe IspH catalysis and the oxidation of the reduced MV

4 Bioinorganic Chemistry and Applications

Table 1 Kinetic parameters of IspH proteins

Source Reducing system 119860410119860280

Kinetics parametersReference

119896cat (minminus1)b SA(120583molminminus1mgminus1) 119870

119872(120583M) 119896cat119870119872

(120583Mminus1 minminus1)

E coliNADPHa 04 116 031 15 08 [11]DT-MV 04 604 163 197 307

[21]DT-MDQc 04 1125 304 316 356A aeolicus DT-MV 222 66 590 04 [22]

P falciparum DT-MV 124 21 39 32[23]NADPH 103

GbIspH1-full DT-MV 017 369 67 173 213 This workGbIspH1-truncated 018 347 72 149 233aNADPH was added with flavodoxinflavodoxin reductase or ferredoxinferredoxin-NADP+ reductasebMW was estimated based on the peptide sequence IspH from E coli 370 kDa A aeolicus 318 kDa P falciparum 588 kDac67-dihydro-211-dimethyldipyrido[12-a21-c ]pyrazinium dibromide (MDQ) is a MV analog

(a) (b)

Figure 2 I-TASSER predicted structure of GbIspH1-full (a) is presented with bacterial IspH ((b) 3KE8 in orange) Residues of the conservedCys His and Phe are depicted with ball and stick model and the substrate HMBPP is represented with stick model The extra N-terminalregions (magenta 1ndash61 green 52ndash131) were colored differently

monitored by the electronic absorption decrease is related tothe IspH activity [20]

Substrate-dependent initial velocity curves of GbIspH1-full and GbIspH1-truncated showed typical Michealis-Menten kinetics (Figure S4) and catalytic activities of bothGbIspH1 enzymes were comparable to the other reportedIspH proteins (Table 1) For example IspH from E coliwith the same reducing system showed 119896cat 604minminus1 and119870119872

197 120583M [21] Between GbIspH1-full and GbIspH1-truncated similar kinetic parameters of 119896cat (369 plusmn 10 versus347 plusmn 12minminus1) and 119870

119872(173 plusmn 19 versus 149 plusmn 23 120583M)

were obtained respectively Although it is not distinctivelydifferent GbIspH1-full appears to exhibit better 119896cat andhigher 119870

119872 In the predicted three-dimensional protein

structure of GbIspH1 N-terminal peptide is located near

the substrate binding site (Figure 2) The relatively low119870119872

value obtained from GbIspH1-truncated may be dueto the absence of extra N-terminal peptide chain which ispresumably positioned on top of the substrate binding site

Activity profiles of GbIspH1-full and GbIspH1-truncatedat the range of pH 55ndash10 showed that two GbIspH1 proteinsexhibited maximum activity at different pH values WhereasGbIspH1-full showed the maximum enzyme activity at pH80 GbIspH1-truncated showed the highest activity at pH 75Altincicek et al [22] reported that the maximum activity ofIspH from Aquifex aeolicus was found in the range of pH70ndash75 When the pH range of more than 50 maximumactivity was compared the range of GbIspH1-truncated (pH56ndash93) was much broader than that of GbIspH1-full (pH66ndash91) (Figure 3) Acidic pH region showing 50maximum

Bioinorganic Chemistry and Applications 5

Con

sum

ptio

n of

HM

BPP

(120583M

min

)120

100

80

60

40

20

0

pH5 6 7 8 9 10

pKa166pKa291

(a)

Con

sum

ptio

n of

HM

BPP

(120583M

min

)

120

100

80

60

40

20

0

pH5 6 7 8 9 10

pKa156pKa293

(b)

Figure 3 The pH-dependent activity profiles of GbIspH1-full (a) and GbIspH1-truncated (b)

0 20 40 600

50

100

150

200

250

300

Temperature (1K)

000

30

000

32

000

34

000

36

0005101520253035

Con

sum

ptio

n of

HM

BPP

(120583M

min

)

Temperature (∘C)

ln k

(sminus1)

(a)

0 20 40 600

50

100

150

200

250

300

350

Temperature (1K)

000

30

000

32

000

34

000

36

0005101520253035

Con

sum

ptio

n of

HM

BPP

(120583M

min

)

Temperature (∘C)

ln k

(sminus1)

(b)

Figure 4 Temperature-dependent activity profiles of GbIspH1-full (a) and GbIspH1-truncated (b) with the Arrhenius plot inserts

activity especially was shifted by 10 pH unit in GbIspH1-full Therefore it was proposed that extra N-terminal regionof GbIspH1-full influenced the pH dependency of enzymeactivity Certain amino acid residues in theN-terminal regionmay modulate the proton transfer steps which are requiredduring the catalysis or conformational changes accompaniedduring the IspH catalysis could be perturbed in case ofGbIspH1-truncated by truncating the N-terminal region ofGbIspH1-full

To find whether the extra N-terminal peptide region ofGbIspH1 influences the enzyme reaction pathway activationenergy values of GbIspH1-full and GbIspH1-truncated weredetermined by measuring temperature-dependent enzymeactivities at 0ndash50∘C As shown in Figure 4 temperature-dependent reaction rate constants of both enzymes werealmost identical and the activation energy (119864

119886) values

obtained by Arrhenius equation were 365 plusmn 16 and350 plusmn 19 kJmol for GbIspH1-full and GbIspH1-truncated

respectively If the extra N-terminal peptide region ofGbIspH1-full affected rate-limiting reaction step the activa-tion energy values of the reactions catalyzed by GbIspH1-full and GbIspH1-truncated are expected to be significantlydifferent However the result strongly suggested that theextra N-terminal region did not alter the rate-limiting stepof GbIspH1 catalysis The activation energy values obtainedfromGbIspH1-full andGbIspH1-truncatedweremuch higherthan the reported 119864

11988680 kJmol of Plasmodium falciparum

IspH [23] and comparable to the activation energy of 49 plusmn2 kJmol reported for the thermophilic A aeolicus [22]

Although it is not known yet which reaction step inthe IspH enzyme reaction pathway is the rate-limiting stepXiao et al [21] reported that the activity of IspH could besignificantly increased by changing electron donors Fur-thermore the conformational change following the electrontransfer step is generally found as the rate-limiting stepof the redox-active metalloenzyme catalysis For example

6 Bioinorganic Chemistry and Applications

OH

OPP

OPP

OPP

HMBPP

IPP

DMAPP

OPP

OP

O

O

O

H

P

O

Fe

O

OPP

HFe

ab

a

b

Allylic anion intermediate

H2O

H2O

eminusH+ eminus

minus

Fe4S4Fe4S4

minusO

Ominus

∙

2eminusH+

Figure 5 Proposed IspH-catalyzed reaction mechanism of isopentenyl diphosphate biosynthesis Steps a and b represent possible protontransfer pathways to the allylic anion intermediate which results in different product formation

conformational change of MoFe protein required to separatedocked Fe protein after electron transfer is the rate-limitingstep of nitrogenase catalysisTherefore it is very likely that thereaction step involving separation of the biological electrondonor from IspH would be rate-limiting step Based on theactivation energy comparison it is also proposed that the N-terminal region of GbIspH1 does not actively participate inthe electron transfer step

33 Reaction Products Identification In plant cell bothMVApathway and MEP pathway are found in cytosol and chloro-plast respectively and believed to have different physiologicalfunctions [24] It is also known that MVA pathway producesonly IPP butMEP pathway provides IPP andDMAPP Differ-ent ratio of IPPDMAPP is required for the specific isoprenyldiphosphate biosynthesis in cytosol and chloroplast andapparentlymore IPP thanDMAPP is required for the synthe-sis of geranyl diphosphate (GPP) farnesyl diphosphate (FPP)and so on Even though short chain prenyl diphosphateincluding IPP (C5) DMAPP (C5) GPP (C10) and FPP (C15)can be transported to other cell organelles in plant cell [25]isoprenyl diphosphate isomerase (IDI) is universally foundfrom the cell organelles including chloroplast mitochondriaand peroxisome Hence maintaining the optimummetabolicflux of IPPDMAPP in the cell looks critical [26] Thisbackground information motivated us to study the productsratio of IPPDMAPP produced by IspH

Interestingly the ratio of IPPDMAPP was also recog-nized as an important trait related with the reaction mech-anism According to the proposed IspH-catalyzed reactionmechanisms the allylic anion species is commonly formedby two different IspH reaction mechanism either from thereduction of substrate allylic radical species [7] or from theorganometallic complex formation between substrate andthe Fe

4S4cluster [8] (Figure 5) The allylic anion species

was suggested as the key intermediate by both mechanismsthat controls product formation and therefore determinesthe products ratio Proton transfer from the intermediate

diphosphate group to the allylic anion moiety is the keyreaction step to control the product distribution of IPPand DMAPP So far all IspH proteins including P falci-parum IspH were reported to produce 45ndash63 1 ratio ofIPPDMAPP [27] Recently we have reported that the IDI-missing plant pathogenic bacterium Burkholderia glumaeproduced 2 1 ratio of IPP and DMAPP [28]

Identification of IPP and DMAPP produced by GbIspH1-full and GbIspH1-truncated was achieved by GCMS anal-ysis of the dephosphorylated products of IPP and DMAPP(Figure S5) and the products ratio was obtained by GC-FIDquantitation (Supplementary Material) Controlled experi-ment with mixtures of standard IPP and DMAPP foundthat the recovery efficiency of prenyl and isoprenyl alcoholswas 1 092 and the experimental results were correctedaccordingly Surprisingly the ratios of IPPDMAPP from thereaction mixtures of GbIspH1-full and GbIspH1-truncatedwere both found as 15 1 reproducibly Because the productsratios obtained from GbIspH1 proteins were significantlylarger than the reported values from other IspH proteins itwas proposed that the proton transfer to the allylic anionintermediate during the GbIspH1 catalysis could be differentfrom the bacterial IspH system

4 Conclusions

In summary catalytically active GbIspH1 proteins ofGbIspH1-full and GbIspH1-truncated were successfullypurified and characterized under anaerobic conditions Theisolated GbIspH1 proteins contained the Fe

4S4cluster like

bacterial IspH and it showed comparable catalytic activityto the other IspH proteins The extra N-terminal region ofGbIspH1 common to plant IspH proteins appears not toinfluence the rate-limiting step because GbIspH1-full andGbIspH1-truncated showed similar kinetic parameters of119896cat and activation energy However the extra N-terminalregion was suggested to modulate the enzyme activity in

Bioinorganic Chemistry and Applications 7

a pH-dependent manner based on the pH-dependentactivity profiles of GbIspH1-full and GbIspH1-truncated

GbIspH1-full with complete GbIspH1 polypeptide wasmeasured to have a maximum enzyme activity at pH 80 andIspH from A aeolicus was reported to show the maximumenzyme activity in the range of pH 70ndash75 [22] Interestinglyspinach IspG another plant MEP pathway enzyme found inchloroplast was reported to accept electrons from photosyn-thetic light reaction [29] and the expression of GbIspH1 wasenhanced by light triggering [18] With all of these reports itis likely that photosynthetic regulation is operating in plantMEP pathway During the photosynthetic light reactionproton is transported from chloroplast stroma to thylakoidlumen and the pH value of stroma approaches pH 80[30] From our pH-dependent activity study it is plausiblethat the N-terminal polypeptide region of GbIspH1 couldmodulate the enzyme activity in a pH-dependent manner inchloroplast

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This work was supported under the framework of Interna-tional Cooperation Program managed by National ResearchFoundation of Korea (NRF-2013K2A1B7A01044254) andby Basic Science Research Program through the NationalResearch Foundation of Korea (NRF) funded by theMinistryof Education (NRF-2010-0020984) The authors also thankto Dr Sun Hee Kim at KBSI for the preliminary EPRmeasurements

References

[1] M Rohmer M Knani P Simonin B Sutter and H SahmldquoIsoprenoid biosynthesis in bacteria a novel pathway for theearly steps leading to isopentenyl diphosphaterdquo BiochemicalJournal vol 295 no 2 pp 517ndash524 1993

[2] T Kuzuyama and H Seto ldquoTwo distinct pathways for essentialmetabolic precursors for isoprenoid biosynthesisrdquo Proceedingsof the Japan Academy Series B Physical and Biological Sciencesvol 88 no 3 pp 41ndash52 2012

[3] W Eisenreich A Bacher D Arigoni and F Rohdich ldquoBiosyn-thesis of isoprenoids via the non-mevalonate pathwayrdquo Cellularand Molecular Life Sciences vol 61 no 12 pp 1401ndash1426 2004

[4] P Adam S Hecht W Eisenreich et al ldquoBiosynthesis of ter-penes studies on 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphos-phate reductaserdquo Proceedings of the National Academy of Sci-ences of the United States of America vol 99 no 19 pp 12108ndash12113 2002

[5] F Rohdich S Hecht A Bacher and W Eisenreich ldquoDeoxyxy-lulose phosphate pathway of isoprenoid biosynthesis Discoveryand function of ispDEFGH genes and their cognate enzymesrdquoPure and Applied Chemistry vol 75 no 2-3 pp 393ndash405 2003

[6] M Seemann K Janthawornpong J Schweizer et al ldquoIso-prenoid biosynthesis via the MEP pathway in vivo Mossbauer

spectroscopy identifies a [4Fe-4S]2+ center with unusual coor-dination sphere in the LytB proteinrdquo Journal of the AmericanChemical Society vol 131 no 37 pp 13184ndash13185 2009

[7] T Grawert F Rohdich L Span et al ldquoStructure of active IspHenzyme fromescherichia coli providesmechanistic insights intosubstrate reductionrdquoAngewandte Chemie International Editionvol 48 no 31 pp 5756ndash5759 2009

[8] T Grawert I Span W Eisenreich et al ldquoProbing the reactionmechanism of IspH protein by X-ray structure analysisrdquo Pro-ceedings of the National Academy of Sciences of the United Statesof America vol 107 no 3 pp 1077ndash1081 2010

[9] I Rekittke J Wiesner R Rohrich et al ldquoStructure of (E)-4-hydroxy-3-methyl-but-2-enyl diphosphate reductase the ter-minal enzyme of the non-mevalonate pathwayrdquo Journal of theAmerican Chemical Society vol 130 no 51 pp 17206ndash172072008

[10] F Rohdich F Zepeck P Adam et al ldquoThe deoxyxylulosephosphate pathway of isoprenoid biosynthesis studies on themechanisms of the reactions catalyzed by IspG and IspHproteinrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 100 no 4 pp 1586ndash1591 2003

[11] Y Xiao Z K Zhao and P Liu ldquoMechanistic studies of IspHin the deoxyxylulose phosphate pathway heterolytic C-O bondcleavage at C4 positionrdquo Journal of the American ChemicalSociety vol 130 no 7 pp 2164ndash2165 2008

[12] W Wang K Wang Y-L Liu et al ldquoBioorganometallic mech-anism of action and inhibition of IspHrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 107 no 10 pp 4522ndash4527 2010

[13] I Span K Wang W Wang et al ldquoDiscovery of acetylenehydratase activity of the iron-sulphur protein IspHrdquo NatureCommunications vol 3 article 1042 2012

[14] S Hecht S Amslinger J Jauch et al ldquoStudies on the non-mevalonate isoprenoid biosynthetic pathway Simple methodsfor preparation of isotope-labeled (E)-1-hydroxy-2-methylbut-2-enyl 4-diphosphaterdquo Tetrahedron Letters vol 43 no 49 pp8929ndash8933 2002

[15] M J A Bailey and J W Noble ldquoQuantitation of proteinrdquoMethods in Enzymology vol 463 pp 73ndash95 2009

[16] M C Kennedy T A Kent M Emptage H Merkle H Beinertand E Munck ldquoEvidence for the formation of a linear [3Fe-4S] cluster in partially unfolded aconitaserdquo Journal of BiologicalChemistry vol 259 no 23 pp 14463ndash14471 1984

[17] F Tan Y Zhang J Wang J Wei Y Cai and X Qian ldquoAnefficient method for dephosphorylation of phosphopeptides bycerium oxiderdquo Journal of Mass Spectrometry vol 43 no 5 pp628ndash632 2008

[18] S-M Kim T Kuzuyama A Kobayashi T Sando Y-JChang and S-U Kim ldquo1-Hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase (IDS) is encoded by multicopy genes ingymnosperms Ginkgo biloba and Pinus taedardquo Planta vol 227no 2 pp 287ndash298 2008

[19] W Xu N S Lees D Hall D Welideniya B M Hoffman andE C Duin ldquoA closer look at the spectroscopic properties ofpossible reaction intermediates in wild-type and mutant (E)-4-hydroxy-3-methylbut-2-enyl diphosphate reductaserdquo Biochem-istry vol 51 no 24 pp 4835ndash4849 2012

[20] H S Kim B-K Shin and J Han ldquoHigh-throughput HDRinhibitor screeningrdquo Journal of the Korean Society for AppliedBiological Chemistry vol 57 no 1 pp 67ndash72 2014

[21] Y Xiao L Chu Y Sanakis and P Liu ldquoRevisiting the IspHcatalytic system in the deoxyxylulose phosphate pathway

8 Bioinorganic Chemistry and Applications

achieving high activityrdquo Journal of the American ChemicalSociety vol 131 no 29 pp 9931ndash9933 2009

[22] B Altincicek E C Duin A Reichenberg et al ldquoLytB proteincatalyzes the terminal step of the 2-C-methyl-D-erythritol-4-phosphate pathway of isoprenoid biosynthesisrdquo FEBS Lettersvol 532 no 3 pp 437ndash440 2002

[23] R C Rohrich N Englert K Troschke et al ldquoReconstitutionof an apicoplast-localised electron transfer pathway involved inthe isoprenoid biosynthesis of Plasmodium falciparumrdquo FEBSLetters vol 579 no 28 pp 6433ndash6438 2005

[24] M Rodrıguez-Concepcion ldquoEarly steps in isoprenoid biosyn-thesis multilevel regulation of the supply of common precur-sors in plant cellsrdquo Phytochemistry Reviews vol 5 no 1 pp 1ndash152006

[25] J A Bick and B M Lange ldquoMetabolic cross talk betweencytosolic and plastidial pathways of isoprenoid biosynthesisunidirectional transport of intermediates across the chloroplastenvelope membranerdquo Archives of Biochemistry and Biophysicsvol 415 no 2 pp 146ndash154 2003

[26] P Pulido C Perello and M Rodrıguez-Concepcion ldquoNewinsights into plant isoprenoidmetabolismrdquoMolecular Plant vol5 no 5 pp 964ndash967 2012

[27] T Grawert J Kaiser F Zepeck et al ldquoIspH protein ofEscherichia coli studies on iron-sulfur cluster implementationand catalysisrdquo Journal of theAmericanChemical Society vol 126no 40 pp 12847ndash12855 2004

[28] M Kwon B-K Shin J Lee J Han and S-U Kim ldquoCharacter-ization of Burkholderia glumae BGR1 4-hydroxy-3-methylbut-2-enyl diphosphate reductase (HDR) the terminal enzyme in2-C-methyl-d-erythritol 4-phosphate (MEP) pathwayrdquo Journalof the Korean Society for Applied Biological Chemistry vol 56no 1 pp 35ndash40 2013

[29] M Seemann B Tse Sum Bui M Wolff M Miginiac-Maslowand M Rohmer ldquoIsoprenoid biosynthesis in plant chloroplastsvia the MEP pathway direct thylakoidferredoxin-dependentphotoreduction of GcpEIspGrdquo FEBS Letters vol 580 no 6 pp1547ndash1552 2006

[30] M Hauser H Eichelmann V Oja U Heber and A LaiskldquoStimulation by light of rapid pH regulation in the chloroplaststroma in vivo as indicated by CO

2solubilization in leavesrdquo

Plant Physiology vol 108 no 3 pp 1059ndash1066 1995

Bioinorganic Chemistry and Applications 3

initiate the reaction while the required reaction temperaturewas maintained

24 Reaction Product Identification To isolate reaction prod-ucts enzyme reaction was carried out with GbIspH1 (1120583M)HMBPP (53mM)MV (50mM) andDT (75mM) in 300 120583Lof 50mM HEPES buffer (pH 80) at room temperature for3 h under anaerobic atmosphere After reaction ceriumoxide(075 g) in 300 120583L 02N ammonia solution was added to theenzyme reaction mixture [17] The solution was reacted for1 h at 40∘C with mild stirring and the dephosphorylatedproducts were extracted with ethyl acetate (200120583L times 3) ForGCMS analysis Agilent 7890A (GC) equipped with DB-5MS (19091S-433) column (30m 025mm and 025 120583m) wasconnected with Agilent 5975C (MSD) detector The sample(1 120583L) was injected to the injector (250∘C) in splitless modeTemperature of oven was kept at 40∘C for 5min beforeincreasing to 450∘C (10∘Cmin) Mass spectra were obtainedwith positive ionization mode by electron ionization (70 eV)

25 Sequence Analysis and Protein Structure PredictionAmino acid sequences of IspH proteins were aligned usingthe MultAlin program (httpmultalintoulouseinrafrmultalinmultalinhtml) Molecular weight and molarextinction coefficient of GbIspH1-full and GbIspH1-trun-cated were calculated using Protein Calculator v33 program(httpwwwscrippsedusimcdputnamprotcalchtml) Predic-tion of 3-dimensional protein structures of GbIspH1-full wasachieved by I-TASSER (httpzhanglabccmbmedumicheduI-TASSER) server

3 Results and Discussion

FeS proteins participate in many important biologicalprocesses such as photosynthesis and energy metabolismBesides the electron transfer FeS enzymes catalyze manyunique reactions of nitrogen fixation hydrogen productionCO oxidation and more Even simple cuboidal [4Fe

4S]

cluster acts as a cofactor in the radical SAMreaction and IspHreduction

Whereas extensive biochemical characterization of bac-terial IspH has been reported no enzyme kinetics on plantIspH are available Here we first reported catalytic propertiesof GbIspH1 Kim et al [18] suggested that the extra N-terminal peptide region contained the 10ndash40 residue-longtransit peptides whichmay facilitate the translocation of IspHinto chloroplast However biochemical function of the otherN-terminal polypeptide region between the transit peptideand catalytic domain is not clear yetThepurpose of this studywas to study the difference between plant IspH and bacterialIspH proteins and to investigate the possible function of theextra N-terminal polypeptide region of GbIspH1 The N-terminal truncated GbIspH1 used in this study was preparedby removing 61 amino acids at the N-terminus of GbIspH1

31 Purification and Characterization of GbIspH1 GbIspH1proteins of GbIspH1-full and GbIspH1-truncated were puri-fied by Ni-NTA affinity column chromatography and

the purity of proteins was confirmed by SDS-PAGE (FigureS2a) The apparent molecular weights of GbIspH1-full andGbIspH1-truncated estimated from the band positions were566 and 493 kDa which were close to the theoretical valuesof 560 and 497 kDa respectively UV-Vis spectra of thepurified GbIspH1-full and GbIspH1-truncated were obtainedunder anaerobic conditions and the absorptions at 410 nmA410

characteristic to the Fe4S4cluster were observed for

both (Figure S2b)TheA410

A280

ratio of the [Fe4S4]2+ cluster-

containing protein measured by UV-Vis spectrophotometerhas been used to evaluate the percentage of holoprotein in thepurified metalloproteinThe A

410A280

ratios of GbIspH1-fulland GbIspH1-truncated were measured to be 018 and 017respectively and both proteins did not require reconstitutionof the Fe

4S4cluster Iron contents of GbIspH1-full and

GbIspH1-truncated were also determined by colorimetricmethod using ferene and 31 plusmn 02 and 35 plusmn 03 Feproteinwere determined for GbIspH1-full and GbIspH1-truncatedrespectively The incorporation of the Fe

4S4cluster in the

purified GbIspH1 proteins was finally confirmed by EPRspectroscopy The methyl viologen- (MV-) reduced WFTshowed rhombic signals at 20K characteristic to the reduced[Fe4S4]+ cluster (Figure S3) CD spectra obtained from

GbIspH1-full and GbIspH1-truncated were very similar andindicated that both proteins were folded similarly (FigureS2c)TheCD spectra were also similar to those reported fromAaIspH [19] suggesting that IspH proteins from plant andbacteria are similar in their secondary structure composi-tions

The purification of GbIspH1-full and GbIspH1-truncatedproteins was carried out under oxygen-free conditions andthe identification of the FeS cluster required a few differentbiophysical characterizationsThe isolated proteins exhibitedpale brown color and the existence of the [Fe

4S4]2+ cluster

was evident from the electronic spectra With protein quan-titation and Fe analysis incorporation of the [Fe

4S4] cluster

in GbIspH1-full and GbIspH1-truncated was confirmed EPRspectroscopic study of the as-isolated and dithionite- (DT-) reduced proteins supported that the [Fe

4S4] cluster in

GbIspH1 shuttled +2 and +1 redox state

32 Enzyme Kinetics of GbIspH1 To measure the enzymeactivity of the purified IspH electrons from the elec-tron donor are required because IspH catalyzes the reduc-tive dehydroxylation of HMBPP A few in vitro elec-tron donors including biological NADPHflavodoxin reduc-taseflavodoxin and abiological DTMV reducing systemshave been utilized for the steady-state kinetics of IspH [11]In this study DT-reduced MV was used as an electrondonor and substrate-dependent reaction rates of GbIspH1-full and GbIspH1-truncated were measured in the presenceof HMBPP by spectrophotometry In detail one-electronreduced MV exhibits two strong electronic absorptionsat 732 nm (120576

732= 3150Mminus1 cmminus1) and 604 nm (120576

604=

18750Mminus1 cmminus1) and two molecules of reduced MV areconsumed for the product formation by IspH The elec-tron transfer and HMBPP reduction are tightly coupled inthe IspH catalysis and the oxidation of the reduced MV

4 Bioinorganic Chemistry and Applications

Table 1 Kinetic parameters of IspH proteins

Source Reducing system 119860410119860280

Kinetics parametersReference

119896cat (minminus1)b SA(120583molminminus1mgminus1) 119870

119872(120583M) 119896cat119870119872

(120583Mminus1 minminus1)

E coliNADPHa 04 116 031 15 08 [11]DT-MV 04 604 163 197 307

[21]DT-MDQc 04 1125 304 316 356A aeolicus DT-MV 222 66 590 04 [22]

P falciparum DT-MV 124 21 39 32[23]NADPH 103

GbIspH1-full DT-MV 017 369 67 173 213 This workGbIspH1-truncated 018 347 72 149 233aNADPH was added with flavodoxinflavodoxin reductase or ferredoxinferredoxin-NADP+ reductasebMW was estimated based on the peptide sequence IspH from E coli 370 kDa A aeolicus 318 kDa P falciparum 588 kDac67-dihydro-211-dimethyldipyrido[12-a21-c ]pyrazinium dibromide (MDQ) is a MV analog

(a) (b)

Figure 2 I-TASSER predicted structure of GbIspH1-full (a) is presented with bacterial IspH ((b) 3KE8 in orange) Residues of the conservedCys His and Phe are depicted with ball and stick model and the substrate HMBPP is represented with stick model The extra N-terminalregions (magenta 1ndash61 green 52ndash131) were colored differently

monitored by the electronic absorption decrease is related tothe IspH activity [20]

Substrate-dependent initial velocity curves of GbIspH1-full and GbIspH1-truncated showed typical Michealis-Menten kinetics (Figure S4) and catalytic activities of bothGbIspH1 enzymes were comparable to the other reportedIspH proteins (Table 1) For example IspH from E coliwith the same reducing system showed 119896cat 604minminus1 and119870119872

197 120583M [21] Between GbIspH1-full and GbIspH1-truncated similar kinetic parameters of 119896cat (369 plusmn 10 versus347 plusmn 12minminus1) and 119870

119872(173 plusmn 19 versus 149 plusmn 23 120583M)

were obtained respectively Although it is not distinctivelydifferent GbIspH1-full appears to exhibit better 119896cat andhigher 119870

119872 In the predicted three-dimensional protein

structure of GbIspH1 N-terminal peptide is located near

the substrate binding site (Figure 2) The relatively low119870119872

value obtained from GbIspH1-truncated may be dueto the absence of extra N-terminal peptide chain which ispresumably positioned on top of the substrate binding site

Activity profiles of GbIspH1-full and GbIspH1-truncatedat the range of pH 55ndash10 showed that two GbIspH1 proteinsexhibited maximum activity at different pH values WhereasGbIspH1-full showed the maximum enzyme activity at pH80 GbIspH1-truncated showed the highest activity at pH 75Altincicek et al [22] reported that the maximum activity ofIspH from Aquifex aeolicus was found in the range of pH70ndash75 When the pH range of more than 50 maximumactivity was compared the range of GbIspH1-truncated (pH56ndash93) was much broader than that of GbIspH1-full (pH66ndash91) (Figure 3) Acidic pH region showing 50maximum

Bioinorganic Chemistry and Applications 5

Con

sum

ptio

n of

HM

BPP

(120583M

min

)120

100

80

60

40

20

0

pH5 6 7 8 9 10

pKa166pKa291

(a)

Con

sum

ptio

n of

HM

BPP

(120583M

min

)

120

100

80

60

40

20

0

pH5 6 7 8 9 10

pKa156pKa293

(b)

Figure 3 The pH-dependent activity profiles of GbIspH1-full (a) and GbIspH1-truncated (b)

0 20 40 600

50

100

150

200

250

300

Temperature (1K)

000

30

000

32

000

34

000

36

0005101520253035

Con

sum

ptio

n of

HM

BPP

(120583M

min

)

Temperature (∘C)

ln k

(sminus1)

(a)

0 20 40 600

50

100

150

200

250

300

350

Temperature (1K)

000

30

000

32

000

34

000

36

0005101520253035

Con

sum

ptio

n of

HM

BPP

(120583M

min

)

Temperature (∘C)

ln k

(sminus1)

(b)

Figure 4 Temperature-dependent activity profiles of GbIspH1-full (a) and GbIspH1-truncated (b) with the Arrhenius plot inserts

activity especially was shifted by 10 pH unit in GbIspH1-full Therefore it was proposed that extra N-terminal regionof GbIspH1-full influenced the pH dependency of enzymeactivity Certain amino acid residues in theN-terminal regionmay modulate the proton transfer steps which are requiredduring the catalysis or conformational changes accompaniedduring the IspH catalysis could be perturbed in case ofGbIspH1-truncated by truncating the N-terminal region ofGbIspH1-full

To find whether the extra N-terminal peptide region ofGbIspH1 influences the enzyme reaction pathway activationenergy values of GbIspH1-full and GbIspH1-truncated weredetermined by measuring temperature-dependent enzymeactivities at 0ndash50∘C As shown in Figure 4 temperature-dependent reaction rate constants of both enzymes werealmost identical and the activation energy (119864

119886) values

obtained by Arrhenius equation were 365 plusmn 16 and350 plusmn 19 kJmol for GbIspH1-full and GbIspH1-truncated

respectively If the extra N-terminal peptide region ofGbIspH1-full affected rate-limiting reaction step the activa-tion energy values of the reactions catalyzed by GbIspH1-full and GbIspH1-truncated are expected to be significantlydifferent However the result strongly suggested that theextra N-terminal region did not alter the rate-limiting stepof GbIspH1 catalysis The activation energy values obtainedfromGbIspH1-full andGbIspH1-truncatedweremuch higherthan the reported 119864

11988680 kJmol of Plasmodium falciparum

IspH [23] and comparable to the activation energy of 49 plusmn2 kJmol reported for the thermophilic A aeolicus [22]

Although it is not known yet which reaction step inthe IspH enzyme reaction pathway is the rate-limiting stepXiao et al [21] reported that the activity of IspH could besignificantly increased by changing electron donors Fur-thermore the conformational change following the electrontransfer step is generally found as the rate-limiting stepof the redox-active metalloenzyme catalysis For example

6 Bioinorganic Chemistry and Applications

OH

OPP

OPP

OPP

HMBPP

IPP

DMAPP

OPP

OP

O

O

O

H

P

O

Fe

O

OPP

HFe

ab

a

b

Allylic anion intermediate

H2O

H2O

eminusH+ eminus

minus

Fe4S4Fe4S4

minusO

Ominus

∙

2eminusH+

Figure 5 Proposed IspH-catalyzed reaction mechanism of isopentenyl diphosphate biosynthesis Steps a and b represent possible protontransfer pathways to the allylic anion intermediate which results in different product formation

conformational change of MoFe protein required to separatedocked Fe protein after electron transfer is the rate-limitingstep of nitrogenase catalysisTherefore it is very likely that thereaction step involving separation of the biological electrondonor from IspH would be rate-limiting step Based on theactivation energy comparison it is also proposed that the N-terminal region of GbIspH1 does not actively participate inthe electron transfer step

33 Reaction Products Identification In plant cell bothMVApathway and MEP pathway are found in cytosol and chloro-plast respectively and believed to have different physiologicalfunctions [24] It is also known that MVA pathway producesonly IPP butMEP pathway provides IPP andDMAPP Differ-ent ratio of IPPDMAPP is required for the specific isoprenyldiphosphate biosynthesis in cytosol and chloroplast andapparentlymore IPP thanDMAPP is required for the synthe-sis of geranyl diphosphate (GPP) farnesyl diphosphate (FPP)and so on Even though short chain prenyl diphosphateincluding IPP (C5) DMAPP (C5) GPP (C10) and FPP (C15)can be transported to other cell organelles in plant cell [25]isoprenyl diphosphate isomerase (IDI) is universally foundfrom the cell organelles including chloroplast mitochondriaand peroxisome Hence maintaining the optimummetabolicflux of IPPDMAPP in the cell looks critical [26] Thisbackground information motivated us to study the productsratio of IPPDMAPP produced by IspH

Interestingly the ratio of IPPDMAPP was also recog-nized as an important trait related with the reaction mech-anism According to the proposed IspH-catalyzed reactionmechanisms the allylic anion species is commonly formedby two different IspH reaction mechanism either from thereduction of substrate allylic radical species [7] or from theorganometallic complex formation between substrate andthe Fe

4S4cluster [8] (Figure 5) The allylic anion species

was suggested as the key intermediate by both mechanismsthat controls product formation and therefore determinesthe products ratio Proton transfer from the intermediate

diphosphate group to the allylic anion moiety is the keyreaction step to control the product distribution of IPPand DMAPP So far all IspH proteins including P falci-parum IspH were reported to produce 45ndash63 1 ratio ofIPPDMAPP [27] Recently we have reported that the IDI-missing plant pathogenic bacterium Burkholderia glumaeproduced 2 1 ratio of IPP and DMAPP [28]

Identification of IPP and DMAPP produced by GbIspH1-full and GbIspH1-truncated was achieved by GCMS anal-ysis of the dephosphorylated products of IPP and DMAPP(Figure S5) and the products ratio was obtained by GC-FIDquantitation (Supplementary Material) Controlled experi-ment with mixtures of standard IPP and DMAPP foundthat the recovery efficiency of prenyl and isoprenyl alcoholswas 1 092 and the experimental results were correctedaccordingly Surprisingly the ratios of IPPDMAPP from thereaction mixtures of GbIspH1-full and GbIspH1-truncatedwere both found as 15 1 reproducibly Because the productsratios obtained from GbIspH1 proteins were significantlylarger than the reported values from other IspH proteins itwas proposed that the proton transfer to the allylic anionintermediate during the GbIspH1 catalysis could be differentfrom the bacterial IspH system

4 Conclusions

In summary catalytically active GbIspH1 proteins ofGbIspH1-full and GbIspH1-truncated were successfullypurified and characterized under anaerobic conditions Theisolated GbIspH1 proteins contained the Fe

4S4cluster like

bacterial IspH and it showed comparable catalytic activityto the other IspH proteins The extra N-terminal region ofGbIspH1 common to plant IspH proteins appears not toinfluence the rate-limiting step because GbIspH1-full andGbIspH1-truncated showed similar kinetic parameters of119896cat and activation energy However the extra N-terminalregion was suggested to modulate the enzyme activity in

Bioinorganic Chemistry and Applications 7

a pH-dependent manner based on the pH-dependentactivity profiles of GbIspH1-full and GbIspH1-truncated

GbIspH1-full with complete GbIspH1 polypeptide wasmeasured to have a maximum enzyme activity at pH 80 andIspH from A aeolicus was reported to show the maximumenzyme activity in the range of pH 70ndash75 [22] Interestinglyspinach IspG another plant MEP pathway enzyme found inchloroplast was reported to accept electrons from photosyn-thetic light reaction [29] and the expression of GbIspH1 wasenhanced by light triggering [18] With all of these reports itis likely that photosynthetic regulation is operating in plantMEP pathway During the photosynthetic light reactionproton is transported from chloroplast stroma to thylakoidlumen and the pH value of stroma approaches pH 80[30] From our pH-dependent activity study it is plausiblethat the N-terminal polypeptide region of GbIspH1 couldmodulate the enzyme activity in a pH-dependent manner inchloroplast

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This work was supported under the framework of Interna-tional Cooperation Program managed by National ResearchFoundation of Korea (NRF-2013K2A1B7A01044254) andby Basic Science Research Program through the NationalResearch Foundation of Korea (NRF) funded by theMinistryof Education (NRF-2010-0020984) The authors also thankto Dr Sun Hee Kim at KBSI for the preliminary EPRmeasurements

References

[1] M Rohmer M Knani P Simonin B Sutter and H SahmldquoIsoprenoid biosynthesis in bacteria a novel pathway for theearly steps leading to isopentenyl diphosphaterdquo BiochemicalJournal vol 295 no 2 pp 517ndash524 1993

[2] T Kuzuyama and H Seto ldquoTwo distinct pathways for essentialmetabolic precursors for isoprenoid biosynthesisrdquo Proceedingsof the Japan Academy Series B Physical and Biological Sciencesvol 88 no 3 pp 41ndash52 2012

[3] W Eisenreich A Bacher D Arigoni and F Rohdich ldquoBiosyn-thesis of isoprenoids via the non-mevalonate pathwayrdquo Cellularand Molecular Life Sciences vol 61 no 12 pp 1401ndash1426 2004

[4] P Adam S Hecht W Eisenreich et al ldquoBiosynthesis of ter-penes studies on 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphos-phate reductaserdquo Proceedings of the National Academy of Sci-ences of the United States of America vol 99 no 19 pp 12108ndash12113 2002

[5] F Rohdich S Hecht A Bacher and W Eisenreich ldquoDeoxyxy-lulose phosphate pathway of isoprenoid biosynthesis Discoveryand function of ispDEFGH genes and their cognate enzymesrdquoPure and Applied Chemistry vol 75 no 2-3 pp 393ndash405 2003

[6] M Seemann K Janthawornpong J Schweizer et al ldquoIso-prenoid biosynthesis via the MEP pathway in vivo Mossbauer

spectroscopy identifies a [4Fe-4S]2+ center with unusual coor-dination sphere in the LytB proteinrdquo Journal of the AmericanChemical Society vol 131 no 37 pp 13184ndash13185 2009

[7] T Grawert F Rohdich L Span et al ldquoStructure of active IspHenzyme fromescherichia coli providesmechanistic insights intosubstrate reductionrdquoAngewandte Chemie International Editionvol 48 no 31 pp 5756ndash5759 2009

[8] T Grawert I Span W Eisenreich et al ldquoProbing the reactionmechanism of IspH protein by X-ray structure analysisrdquo Pro-ceedings of the National Academy of Sciences of the United Statesof America vol 107 no 3 pp 1077ndash1081 2010

[9] I Rekittke J Wiesner R Rohrich et al ldquoStructure of (E)-4-hydroxy-3-methyl-but-2-enyl diphosphate reductase the ter-minal enzyme of the non-mevalonate pathwayrdquo Journal of theAmerican Chemical Society vol 130 no 51 pp 17206ndash172072008

[10] F Rohdich F Zepeck P Adam et al ldquoThe deoxyxylulosephosphate pathway of isoprenoid biosynthesis studies on themechanisms of the reactions catalyzed by IspG and IspHproteinrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 100 no 4 pp 1586ndash1591 2003

[11] Y Xiao Z K Zhao and P Liu ldquoMechanistic studies of IspHin the deoxyxylulose phosphate pathway heterolytic C-O bondcleavage at C4 positionrdquo Journal of the American ChemicalSociety vol 130 no 7 pp 2164ndash2165 2008

[12] W Wang K Wang Y-L Liu et al ldquoBioorganometallic mech-anism of action and inhibition of IspHrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 107 no 10 pp 4522ndash4527 2010

[13] I Span K Wang W Wang et al ldquoDiscovery of acetylenehydratase activity of the iron-sulphur protein IspHrdquo NatureCommunications vol 3 article 1042 2012

[14] S Hecht S Amslinger J Jauch et al ldquoStudies on the non-mevalonate isoprenoid biosynthetic pathway Simple methodsfor preparation of isotope-labeled (E)-1-hydroxy-2-methylbut-2-enyl 4-diphosphaterdquo Tetrahedron Letters vol 43 no 49 pp8929ndash8933 2002

[15] M J A Bailey and J W Noble ldquoQuantitation of proteinrdquoMethods in Enzymology vol 463 pp 73ndash95 2009

[16] M C Kennedy T A Kent M Emptage H Merkle H Beinertand E Munck ldquoEvidence for the formation of a linear [3Fe-4S] cluster in partially unfolded aconitaserdquo Journal of BiologicalChemistry vol 259 no 23 pp 14463ndash14471 1984

[17] F Tan Y Zhang J Wang J Wei Y Cai and X Qian ldquoAnefficient method for dephosphorylation of phosphopeptides bycerium oxiderdquo Journal of Mass Spectrometry vol 43 no 5 pp628ndash632 2008

[18] S-M Kim T Kuzuyama A Kobayashi T Sando Y-JChang and S-U Kim ldquo1-Hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase (IDS) is encoded by multicopy genes ingymnosperms Ginkgo biloba and Pinus taedardquo Planta vol 227no 2 pp 287ndash298 2008

[19] W Xu N S Lees D Hall D Welideniya B M Hoffman andE C Duin ldquoA closer look at the spectroscopic properties ofpossible reaction intermediates in wild-type and mutant (E)-4-hydroxy-3-methylbut-2-enyl diphosphate reductaserdquo Biochem-istry vol 51 no 24 pp 4835ndash4849 2012

[20] H S Kim B-K Shin and J Han ldquoHigh-throughput HDRinhibitor screeningrdquo Journal of the Korean Society for AppliedBiological Chemistry vol 57 no 1 pp 67ndash72 2014

[21] Y Xiao L Chu Y Sanakis and P Liu ldquoRevisiting the IspHcatalytic system in the deoxyxylulose phosphate pathway

8 Bioinorganic Chemistry and Applications

achieving high activityrdquo Journal of the American ChemicalSociety vol 131 no 29 pp 9931ndash9933 2009

[22] B Altincicek E C Duin A Reichenberg et al ldquoLytB proteincatalyzes the terminal step of the 2-C-methyl-D-erythritol-4-phosphate pathway of isoprenoid biosynthesisrdquo FEBS Lettersvol 532 no 3 pp 437ndash440 2002

[23] R C Rohrich N Englert K Troschke et al ldquoReconstitutionof an apicoplast-localised electron transfer pathway involved inthe isoprenoid biosynthesis of Plasmodium falciparumrdquo FEBSLetters vol 579 no 28 pp 6433ndash6438 2005

[24] M Rodrıguez-Concepcion ldquoEarly steps in isoprenoid biosyn-thesis multilevel regulation of the supply of common precur-sors in plant cellsrdquo Phytochemistry Reviews vol 5 no 1 pp 1ndash152006

[25] J A Bick and B M Lange ldquoMetabolic cross talk betweencytosolic and plastidial pathways of isoprenoid biosynthesisunidirectional transport of intermediates across the chloroplastenvelope membranerdquo Archives of Biochemistry and Biophysicsvol 415 no 2 pp 146ndash154 2003

[26] P Pulido C Perello and M Rodrıguez-Concepcion ldquoNewinsights into plant isoprenoidmetabolismrdquoMolecular Plant vol5 no 5 pp 964ndash967 2012

[27] T Grawert J Kaiser F Zepeck et al ldquoIspH protein ofEscherichia coli studies on iron-sulfur cluster implementationand catalysisrdquo Journal of theAmericanChemical Society vol 126no 40 pp 12847ndash12855 2004

[28] M Kwon B-K Shin J Lee J Han and S-U Kim ldquoCharacter-ization of Burkholderia glumae BGR1 4-hydroxy-3-methylbut-2-enyl diphosphate reductase (HDR) the terminal enzyme in2-C-methyl-d-erythritol 4-phosphate (MEP) pathwayrdquo Journalof the Korean Society for Applied Biological Chemistry vol 56no 1 pp 35ndash40 2013

[29] M Seemann B Tse Sum Bui M Wolff M Miginiac-Maslowand M Rohmer ldquoIsoprenoid biosynthesis in plant chloroplastsvia the MEP pathway direct thylakoidferredoxin-dependentphotoreduction of GcpEIspGrdquo FEBS Letters vol 580 no 6 pp1547ndash1552 2006

[30] M Hauser H Eichelmann V Oja U Heber and A LaiskldquoStimulation by light of rapid pH regulation in the chloroplaststroma in vivo as indicated by CO

2solubilization in leavesrdquo

Plant Physiology vol 108 no 3 pp 1059ndash1066 1995

4 Bioinorganic Chemistry and Applications

Table 1 Kinetic parameters of IspH proteins

Source Reducing system 119860410119860280

Kinetics parametersReference

119896cat (minminus1)b SA(120583molminminus1mgminus1) 119870

119872(120583M) 119896cat119870119872

(120583Mminus1 minminus1)

E coliNADPHa 04 116 031 15 08 [11]DT-MV 04 604 163 197 307

[21]DT-MDQc 04 1125 304 316 356A aeolicus DT-MV 222 66 590 04 [22]

P falciparum DT-MV 124 21 39 32[23]NADPH 103

GbIspH1-full DT-MV 017 369 67 173 213 This workGbIspH1-truncated 018 347 72 149 233aNADPH was added with flavodoxinflavodoxin reductase or ferredoxinferredoxin-NADP+ reductasebMW was estimated based on the peptide sequence IspH from E coli 370 kDa A aeolicus 318 kDa P falciparum 588 kDac67-dihydro-211-dimethyldipyrido[12-a21-c ]pyrazinium dibromide (MDQ) is a MV analog

(a) (b)

Figure 2 I-TASSER predicted structure of GbIspH1-full (a) is presented with bacterial IspH ((b) 3KE8 in orange) Residues of the conservedCys His and Phe are depicted with ball and stick model and the substrate HMBPP is represented with stick model The extra N-terminalregions (magenta 1ndash61 green 52ndash131) were colored differently

monitored by the electronic absorption decrease is related tothe IspH activity [20]

Substrate-dependent initial velocity curves of GbIspH1-full and GbIspH1-truncated showed typical Michealis-Menten kinetics (Figure S4) and catalytic activities of bothGbIspH1 enzymes were comparable to the other reportedIspH proteins (Table 1) For example IspH from E coliwith the same reducing system showed 119896cat 604minminus1 and119870119872

197 120583M [21] Between GbIspH1-full and GbIspH1-truncated similar kinetic parameters of 119896cat (369 plusmn 10 versus347 plusmn 12minminus1) and 119870

119872(173 plusmn 19 versus 149 plusmn 23 120583M)

were obtained respectively Although it is not distinctivelydifferent GbIspH1-full appears to exhibit better 119896cat andhigher 119870

119872 In the predicted three-dimensional protein

structure of GbIspH1 N-terminal peptide is located near

the substrate binding site (Figure 2) The relatively low119870119872

value obtained from GbIspH1-truncated may be dueto the absence of extra N-terminal peptide chain which ispresumably positioned on top of the substrate binding site

Activity profiles of GbIspH1-full and GbIspH1-truncatedat the range of pH 55ndash10 showed that two GbIspH1 proteinsexhibited maximum activity at different pH values WhereasGbIspH1-full showed the maximum enzyme activity at pH80 GbIspH1-truncated showed the highest activity at pH 75Altincicek et al [22] reported that the maximum activity ofIspH from Aquifex aeolicus was found in the range of pH70ndash75 When the pH range of more than 50 maximumactivity was compared the range of GbIspH1-truncated (pH56ndash93) was much broader than that of GbIspH1-full (pH66ndash91) (Figure 3) Acidic pH region showing 50maximum

Bioinorganic Chemistry and Applications 5

Con

sum

ptio

n of

HM

BPP

(120583M

min

)120

100

80

60

40

20

0

pH5 6 7 8 9 10

pKa166pKa291

(a)

Con

sum

ptio

n of

HM

BPP

(120583M

min

)

120

100

80

60

40

20

0

pH5 6 7 8 9 10

pKa156pKa293

(b)

Figure 3 The pH-dependent activity profiles of GbIspH1-full (a) and GbIspH1-truncated (b)

0 20 40 600

50

100

150

200

250

300

Temperature (1K)

000

30

000

32

000

34

000

36

0005101520253035

Con

sum

ptio

n of

HM

BPP

(120583M

min

)

Temperature (∘C)

ln k

(sminus1)

(a)

0 20 40 600

50

100

150

200

250

300

350

Temperature (1K)

000

30

000

32

000

34

000

36

0005101520253035

Con

sum

ptio

n of

HM

BPP

(120583M

min

)

Temperature (∘C)

ln k

(sminus1)

(b)

Figure 4 Temperature-dependent activity profiles of GbIspH1-full (a) and GbIspH1-truncated (b) with the Arrhenius plot inserts

activity especially was shifted by 10 pH unit in GbIspH1-full Therefore it was proposed that extra N-terminal regionof GbIspH1-full influenced the pH dependency of enzymeactivity Certain amino acid residues in theN-terminal regionmay modulate the proton transfer steps which are requiredduring the catalysis or conformational changes accompaniedduring the IspH catalysis could be perturbed in case ofGbIspH1-truncated by truncating the N-terminal region ofGbIspH1-full

To find whether the extra N-terminal peptide region ofGbIspH1 influences the enzyme reaction pathway activationenergy values of GbIspH1-full and GbIspH1-truncated weredetermined by measuring temperature-dependent enzymeactivities at 0ndash50∘C As shown in Figure 4 temperature-dependent reaction rate constants of both enzymes werealmost identical and the activation energy (119864

119886) values

obtained by Arrhenius equation were 365 plusmn 16 and350 plusmn 19 kJmol for GbIspH1-full and GbIspH1-truncated

respectively If the extra N-terminal peptide region ofGbIspH1-full affected rate-limiting reaction step the activa-tion energy values of the reactions catalyzed by GbIspH1-full and GbIspH1-truncated are expected to be significantlydifferent However the result strongly suggested that theextra N-terminal region did not alter the rate-limiting stepof GbIspH1 catalysis The activation energy values obtainedfromGbIspH1-full andGbIspH1-truncatedweremuch higherthan the reported 119864

11988680 kJmol of Plasmodium falciparum

IspH [23] and comparable to the activation energy of 49 plusmn2 kJmol reported for the thermophilic A aeolicus [22]

Although it is not known yet which reaction step inthe IspH enzyme reaction pathway is the rate-limiting stepXiao et al [21] reported that the activity of IspH could besignificantly increased by changing electron donors Fur-thermore the conformational change following the electrontransfer step is generally found as the rate-limiting stepof the redox-active metalloenzyme catalysis For example

6 Bioinorganic Chemistry and Applications

OH

OPP

OPP

OPP

HMBPP

IPP

DMAPP

OPP

OP

O

O

O

H

P

O

Fe

O

OPP

HFe

ab

a

b

Allylic anion intermediate

H2O

H2O

eminusH+ eminus

minus

Fe4S4Fe4S4

minusO

Ominus

∙

2eminusH+

Figure 5 Proposed IspH-catalyzed reaction mechanism of isopentenyl diphosphate biosynthesis Steps a and b represent possible protontransfer pathways to the allylic anion intermediate which results in different product formation

conformational change of MoFe protein required to separatedocked Fe protein after electron transfer is the rate-limitingstep of nitrogenase catalysisTherefore it is very likely that thereaction step involving separation of the biological electrondonor from IspH would be rate-limiting step Based on theactivation energy comparison it is also proposed that the N-terminal region of GbIspH1 does not actively participate inthe electron transfer step

33 Reaction Products Identification In plant cell bothMVApathway and MEP pathway are found in cytosol and chloro-plast respectively and believed to have different physiologicalfunctions [24] It is also known that MVA pathway producesonly IPP butMEP pathway provides IPP andDMAPP Differ-ent ratio of IPPDMAPP is required for the specific isoprenyldiphosphate biosynthesis in cytosol and chloroplast andapparentlymore IPP thanDMAPP is required for the synthe-sis of geranyl diphosphate (GPP) farnesyl diphosphate (FPP)and so on Even though short chain prenyl diphosphateincluding IPP (C5) DMAPP (C5) GPP (C10) and FPP (C15)can be transported to other cell organelles in plant cell [25]isoprenyl diphosphate isomerase (IDI) is universally foundfrom the cell organelles including chloroplast mitochondriaand peroxisome Hence maintaining the optimummetabolicflux of IPPDMAPP in the cell looks critical [26] Thisbackground information motivated us to study the productsratio of IPPDMAPP produced by IspH

Interestingly the ratio of IPPDMAPP was also recog-nized as an important trait related with the reaction mech-anism According to the proposed IspH-catalyzed reactionmechanisms the allylic anion species is commonly formedby two different IspH reaction mechanism either from thereduction of substrate allylic radical species [7] or from theorganometallic complex formation between substrate andthe Fe

4S4cluster [8] (Figure 5) The allylic anion species

was suggested as the key intermediate by both mechanismsthat controls product formation and therefore determinesthe products ratio Proton transfer from the intermediate

diphosphate group to the allylic anion moiety is the keyreaction step to control the product distribution of IPPand DMAPP So far all IspH proteins including P falci-parum IspH were reported to produce 45ndash63 1 ratio ofIPPDMAPP [27] Recently we have reported that the IDI-missing plant pathogenic bacterium Burkholderia glumaeproduced 2 1 ratio of IPP and DMAPP [28]

Identification of IPP and DMAPP produced by GbIspH1-full and GbIspH1-truncated was achieved by GCMS anal-ysis of the dephosphorylated products of IPP and DMAPP(Figure S5) and the products ratio was obtained by GC-FIDquantitation (Supplementary Material) Controlled experi-ment with mixtures of standard IPP and DMAPP foundthat the recovery efficiency of prenyl and isoprenyl alcoholswas 1 092 and the experimental results were correctedaccordingly Surprisingly the ratios of IPPDMAPP from thereaction mixtures of GbIspH1-full and GbIspH1-truncatedwere both found as 15 1 reproducibly Because the productsratios obtained from GbIspH1 proteins were significantlylarger than the reported values from other IspH proteins itwas proposed that the proton transfer to the allylic anionintermediate during the GbIspH1 catalysis could be differentfrom the bacterial IspH system

4 Conclusions

In summary catalytically active GbIspH1 proteins ofGbIspH1-full and GbIspH1-truncated were successfullypurified and characterized under anaerobic conditions Theisolated GbIspH1 proteins contained the Fe

4S4cluster like

bacterial IspH and it showed comparable catalytic activityto the other IspH proteins The extra N-terminal region ofGbIspH1 common to plant IspH proteins appears not toinfluence the rate-limiting step because GbIspH1-full andGbIspH1-truncated showed similar kinetic parameters of119896cat and activation energy However the extra N-terminalregion was suggested to modulate the enzyme activity in

Bioinorganic Chemistry and Applications 7

a pH-dependent manner based on the pH-dependentactivity profiles of GbIspH1-full and GbIspH1-truncated

GbIspH1-full with complete GbIspH1 polypeptide wasmeasured to have a maximum enzyme activity at pH 80 andIspH from A aeolicus was reported to show the maximumenzyme activity in the range of pH 70ndash75 [22] Interestinglyspinach IspG another plant MEP pathway enzyme found inchloroplast was reported to accept electrons from photosyn-thetic light reaction [29] and the expression of GbIspH1 wasenhanced by light triggering [18] With all of these reports itis likely that photosynthetic regulation is operating in plantMEP pathway During the photosynthetic light reactionproton is transported from chloroplast stroma to thylakoidlumen and the pH value of stroma approaches pH 80[30] From our pH-dependent activity study it is plausiblethat the N-terminal polypeptide region of GbIspH1 couldmodulate the enzyme activity in a pH-dependent manner inchloroplast

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This work was supported under the framework of Interna-tional Cooperation Program managed by National ResearchFoundation of Korea (NRF-2013K2A1B7A01044254) andby Basic Science Research Program through the NationalResearch Foundation of Korea (NRF) funded by theMinistryof Education (NRF-2010-0020984) The authors also thankto Dr Sun Hee Kim at KBSI for the preliminary EPRmeasurements

References

[1] M Rohmer M Knani P Simonin B Sutter and H SahmldquoIsoprenoid biosynthesis in bacteria a novel pathway for theearly steps leading to isopentenyl diphosphaterdquo BiochemicalJournal vol 295 no 2 pp 517ndash524 1993

[2] T Kuzuyama and H Seto ldquoTwo distinct pathways for essentialmetabolic precursors for isoprenoid biosynthesisrdquo Proceedingsof the Japan Academy Series B Physical and Biological Sciencesvol 88 no 3 pp 41ndash52 2012

[3] W Eisenreich A Bacher D Arigoni and F Rohdich ldquoBiosyn-thesis of isoprenoids via the non-mevalonate pathwayrdquo Cellularand Molecular Life Sciences vol 61 no 12 pp 1401ndash1426 2004

[4] P Adam S Hecht W Eisenreich et al ldquoBiosynthesis of ter-penes studies on 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphos-phate reductaserdquo Proceedings of the National Academy of Sci-ences of the United States of America vol 99 no 19 pp 12108ndash12113 2002

[5] F Rohdich S Hecht A Bacher and W Eisenreich ldquoDeoxyxy-lulose phosphate pathway of isoprenoid biosynthesis Discoveryand function of ispDEFGH genes and their cognate enzymesrdquoPure and Applied Chemistry vol 75 no 2-3 pp 393ndash405 2003

[6] M Seemann K Janthawornpong J Schweizer et al ldquoIso-prenoid biosynthesis via the MEP pathway in vivo Mossbauer

spectroscopy identifies a [4Fe-4S]2+ center with unusual coor-dination sphere in the LytB proteinrdquo Journal of the AmericanChemical Society vol 131 no 37 pp 13184ndash13185 2009

[7] T Grawert F Rohdich L Span et al ldquoStructure of active IspHenzyme fromescherichia coli providesmechanistic insights intosubstrate reductionrdquoAngewandte Chemie International Editionvol 48 no 31 pp 5756ndash5759 2009

[8] T Grawert I Span W Eisenreich et al ldquoProbing the reactionmechanism of IspH protein by X-ray structure analysisrdquo Pro-ceedings of the National Academy of Sciences of the United Statesof America vol 107 no 3 pp 1077ndash1081 2010

[9] I Rekittke J Wiesner R Rohrich et al ldquoStructure of (E)-4-hydroxy-3-methyl-but-2-enyl diphosphate reductase the ter-minal enzyme of the non-mevalonate pathwayrdquo Journal of theAmerican Chemical Society vol 130 no 51 pp 17206ndash172072008

[10] F Rohdich F Zepeck P Adam et al ldquoThe deoxyxylulosephosphate pathway of isoprenoid biosynthesis studies on themechanisms of the reactions catalyzed by IspG and IspHproteinrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 100 no 4 pp 1586ndash1591 2003

[11] Y Xiao Z K Zhao and P Liu ldquoMechanistic studies of IspHin the deoxyxylulose phosphate pathway heterolytic C-O bondcleavage at C4 positionrdquo Journal of the American ChemicalSociety vol 130 no 7 pp 2164ndash2165 2008

[12] W Wang K Wang Y-L Liu et al ldquoBioorganometallic mech-anism of action and inhibition of IspHrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 107 no 10 pp 4522ndash4527 2010

[13] I Span K Wang W Wang et al ldquoDiscovery of acetylenehydratase activity of the iron-sulphur protein IspHrdquo NatureCommunications vol 3 article 1042 2012

[14] S Hecht S Amslinger J Jauch et al ldquoStudies on the non-mevalonate isoprenoid biosynthetic pathway Simple methodsfor preparation of isotope-labeled (E)-1-hydroxy-2-methylbut-2-enyl 4-diphosphaterdquo Tetrahedron Letters vol 43 no 49 pp8929ndash8933 2002

[15] M J A Bailey and J W Noble ldquoQuantitation of proteinrdquoMethods in Enzymology vol 463 pp 73ndash95 2009

[16] M C Kennedy T A Kent M Emptage H Merkle H Beinertand E Munck ldquoEvidence for the formation of a linear [3Fe-4S] cluster in partially unfolded aconitaserdquo Journal of BiologicalChemistry vol 259 no 23 pp 14463ndash14471 1984

[17] F Tan Y Zhang J Wang J Wei Y Cai and X Qian ldquoAnefficient method for dephosphorylation of phosphopeptides bycerium oxiderdquo Journal of Mass Spectrometry vol 43 no 5 pp628ndash632 2008

[18] S-M Kim T Kuzuyama A Kobayashi T Sando Y-JChang and S-U Kim ldquo1-Hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase (IDS) is encoded by multicopy genes ingymnosperms Ginkgo biloba and Pinus taedardquo Planta vol 227no 2 pp 287ndash298 2008

[19] W Xu N S Lees D Hall D Welideniya B M Hoffman andE C Duin ldquoA closer look at the spectroscopic properties ofpossible reaction intermediates in wild-type and mutant (E)-4-hydroxy-3-methylbut-2-enyl diphosphate reductaserdquo Biochem-istry vol 51 no 24 pp 4835ndash4849 2012

[20] H S Kim B-K Shin and J Han ldquoHigh-throughput HDRinhibitor screeningrdquo Journal of the Korean Society for AppliedBiological Chemistry vol 57 no 1 pp 67ndash72 2014

[21] Y Xiao L Chu Y Sanakis and P Liu ldquoRevisiting the IspHcatalytic system in the deoxyxylulose phosphate pathway

8 Bioinorganic Chemistry and Applications

achieving high activityrdquo Journal of the American ChemicalSociety vol 131 no 29 pp 9931ndash9933 2009

[22] B Altincicek E C Duin A Reichenberg et al ldquoLytB proteincatalyzes the terminal step of the 2-C-methyl-D-erythritol-4-phosphate pathway of isoprenoid biosynthesisrdquo FEBS Lettersvol 532 no 3 pp 437ndash440 2002

[23] R C Rohrich N Englert K Troschke et al ldquoReconstitutionof an apicoplast-localised electron transfer pathway involved inthe isoprenoid biosynthesis of Plasmodium falciparumrdquo FEBSLetters vol 579 no 28 pp 6433ndash6438 2005

[24] M Rodrıguez-Concepcion ldquoEarly steps in isoprenoid biosyn-thesis multilevel regulation of the supply of common precur-sors in plant cellsrdquo Phytochemistry Reviews vol 5 no 1 pp 1ndash152006

[25] J A Bick and B M Lange ldquoMetabolic cross talk betweencytosolic and plastidial pathways of isoprenoid biosynthesisunidirectional transport of intermediates across the chloroplastenvelope membranerdquo Archives of Biochemistry and Biophysicsvol 415 no 2 pp 146ndash154 2003

[26] P Pulido C Perello and M Rodrıguez-Concepcion ldquoNewinsights into plant isoprenoidmetabolismrdquoMolecular Plant vol5 no 5 pp 964ndash967 2012

[27] T Grawert J Kaiser F Zepeck et al ldquoIspH protein ofEscherichia coli studies on iron-sulfur cluster implementationand catalysisrdquo Journal of theAmericanChemical Society vol 126no 40 pp 12847ndash12855 2004

[28] M Kwon B-K Shin J Lee J Han and S-U Kim ldquoCharacter-ization of Burkholderia glumae BGR1 4-hydroxy-3-methylbut-2-enyl diphosphate reductase (HDR) the terminal enzyme in2-C-methyl-d-erythritol 4-phosphate (MEP) pathwayrdquo Journalof the Korean Society for Applied Biological Chemistry vol 56no 1 pp 35ndash40 2013

[29] M Seemann B Tse Sum Bui M Wolff M Miginiac-Maslowand M Rohmer ldquoIsoprenoid biosynthesis in plant chloroplastsvia the MEP pathway direct thylakoidferredoxin-dependentphotoreduction of GcpEIspGrdquo FEBS Letters vol 580 no 6 pp1547ndash1552 2006

[30] M Hauser H Eichelmann V Oja U Heber and A LaiskldquoStimulation by light of rapid pH regulation in the chloroplaststroma in vivo as indicated by CO

2solubilization in leavesrdquo

Plant Physiology vol 108 no 3 pp 1059ndash1066 1995

Bioinorganic Chemistry and Applications 5

Con

sum

ptio

n of

HM

BPP

(120583M

min

)120

100

80

60

40

20

0

pH5 6 7 8 9 10

pKa166pKa291

(a)

Con

sum

ptio

n of

HM

BPP

(120583M

min

)

120

100

80

60

40

20

0

pH5 6 7 8 9 10

pKa156pKa293

(b)

Figure 3 The pH-dependent activity profiles of GbIspH1-full (a) and GbIspH1-truncated (b)

0 20 40 600

50

100

150

200

250

300

Temperature (1K)

000

30

000

32

000

34

000

36

0005101520253035

Con

sum

ptio

n of

HM

BPP

(120583M

min

)

Temperature (∘C)

ln k

(sminus1)

(a)

0 20 40 600

50

100

150

200

250

300

350

Temperature (1K)

000

30

000

32

000

34

000

36

0005101520253035

Con

sum

ptio

n of

HM

BPP

(120583M

min

)

Temperature (∘C)

ln k

(sminus1)

(b)

Figure 4 Temperature-dependent activity profiles of GbIspH1-full (a) and GbIspH1-truncated (b) with the Arrhenius plot inserts

activity especially was shifted by 10 pH unit in GbIspH1-full Therefore it was proposed that extra N-terminal regionof GbIspH1-full influenced the pH dependency of enzymeactivity Certain amino acid residues in theN-terminal regionmay modulate the proton transfer steps which are requiredduring the catalysis or conformational changes accompaniedduring the IspH catalysis could be perturbed in case ofGbIspH1-truncated by truncating the N-terminal region ofGbIspH1-full

To find whether the extra N-terminal peptide region ofGbIspH1 influences the enzyme reaction pathway activationenergy values of GbIspH1-full and GbIspH1-truncated weredetermined by measuring temperature-dependent enzymeactivities at 0ndash50∘C As shown in Figure 4 temperature-dependent reaction rate constants of both enzymes werealmost identical and the activation energy (119864

119886) values

obtained by Arrhenius equation were 365 plusmn 16 and350 plusmn 19 kJmol for GbIspH1-full and GbIspH1-truncated

respectively If the extra N-terminal peptide region ofGbIspH1-full affected rate-limiting reaction step the activa-tion energy values of the reactions catalyzed by GbIspH1-full and GbIspH1-truncated are expected to be significantlydifferent However the result strongly suggested that theextra N-terminal region did not alter the rate-limiting stepof GbIspH1 catalysis The activation energy values obtainedfromGbIspH1-full andGbIspH1-truncatedweremuch higherthan the reported 119864

11988680 kJmol of Plasmodium falciparum

IspH [23] and comparable to the activation energy of 49 plusmn2 kJmol reported for the thermophilic A aeolicus [22]

Although it is not known yet which reaction step inthe IspH enzyme reaction pathway is the rate-limiting stepXiao et al [21] reported that the activity of IspH could besignificantly increased by changing electron donors Fur-thermore the conformational change following the electrontransfer step is generally found as the rate-limiting stepof the redox-active metalloenzyme catalysis For example

6 Bioinorganic Chemistry and Applications

OH

OPP

OPP

OPP

HMBPP

IPP

DMAPP

OPP

OP

O

O

O

H

P

O

Fe

O

OPP

HFe

ab

a

b

Allylic anion intermediate

H2O

H2O

eminusH+ eminus

minus

Fe4S4Fe4S4

minusO

Ominus

∙

2eminusH+

Figure 5 Proposed IspH-catalyzed reaction mechanism of isopentenyl diphosphate biosynthesis Steps a and b represent possible protontransfer pathways to the allylic anion intermediate which results in different product formation

conformational change of MoFe protein required to separatedocked Fe protein after electron transfer is the rate-limitingstep of nitrogenase catalysisTherefore it is very likely that thereaction step involving separation of the biological electrondonor from IspH would be rate-limiting step Based on theactivation energy comparison it is also proposed that the N-terminal region of GbIspH1 does not actively participate inthe electron transfer step

33 Reaction Products Identification In plant cell bothMVApathway and MEP pathway are found in cytosol and chloro-plast respectively and believed to have different physiologicalfunctions [24] It is also known that MVA pathway producesonly IPP butMEP pathway provides IPP andDMAPP Differ-ent ratio of IPPDMAPP is required for the specific isoprenyldiphosphate biosynthesis in cytosol and chloroplast andapparentlymore IPP thanDMAPP is required for the synthe-sis of geranyl diphosphate (GPP) farnesyl diphosphate (FPP)and so on Even though short chain prenyl diphosphateincluding IPP (C5) DMAPP (C5) GPP (C10) and FPP (C15)can be transported to other cell organelles in plant cell [25]isoprenyl diphosphate isomerase (IDI) is universally foundfrom the cell organelles including chloroplast mitochondriaand peroxisome Hence maintaining the optimummetabolicflux of IPPDMAPP in the cell looks critical [26] Thisbackground information motivated us to study the productsratio of IPPDMAPP produced by IspH

Interestingly the ratio of IPPDMAPP was also recog-nized as an important trait related with the reaction mech-anism According to the proposed IspH-catalyzed reactionmechanisms the allylic anion species is commonly formedby two different IspH reaction mechanism either from thereduction of substrate allylic radical species [7] or from theorganometallic complex formation between substrate andthe Fe

4S4cluster [8] (Figure 5) The allylic anion species

was suggested as the key intermediate by both mechanismsthat controls product formation and therefore determinesthe products ratio Proton transfer from the intermediate

diphosphate group to the allylic anion moiety is the keyreaction step to control the product distribution of IPPand DMAPP So far all IspH proteins including P falci-parum IspH were reported to produce 45ndash63 1 ratio ofIPPDMAPP [27] Recently we have reported that the IDI-missing plant pathogenic bacterium Burkholderia glumaeproduced 2 1 ratio of IPP and DMAPP [28]

Identification of IPP and DMAPP produced by GbIspH1-full and GbIspH1-truncated was achieved by GCMS anal-ysis of the dephosphorylated products of IPP and DMAPP(Figure S5) and the products ratio was obtained by GC-FIDquantitation (Supplementary Material) Controlled experi-ment with mixtures of standard IPP and DMAPP foundthat the recovery efficiency of prenyl and isoprenyl alcoholswas 1 092 and the experimental results were correctedaccordingly Surprisingly the ratios of IPPDMAPP from thereaction mixtures of GbIspH1-full and GbIspH1-truncatedwere both found as 15 1 reproducibly Because the productsratios obtained from GbIspH1 proteins were significantlylarger than the reported values from other IspH proteins itwas proposed that the proton transfer to the allylic anionintermediate during the GbIspH1 catalysis could be differentfrom the bacterial IspH system

4 Conclusions

In summary catalytically active GbIspH1 proteins ofGbIspH1-full and GbIspH1-truncated were successfullypurified and characterized under anaerobic conditions Theisolated GbIspH1 proteins contained the Fe

4S4cluster like

bacterial IspH and it showed comparable catalytic activityto the other IspH proteins The extra N-terminal region ofGbIspH1 common to plant IspH proteins appears not toinfluence the rate-limiting step because GbIspH1-full andGbIspH1-truncated showed similar kinetic parameters of119896cat and activation energy However the extra N-terminalregion was suggested to modulate the enzyme activity in

Bioinorganic Chemistry and Applications 7

a pH-dependent manner based on the pH-dependentactivity profiles of GbIspH1-full and GbIspH1-truncated

GbIspH1-full with complete GbIspH1 polypeptide wasmeasured to have a maximum enzyme activity at pH 80 andIspH from A aeolicus was reported to show the maximumenzyme activity in the range of pH 70ndash75 [22] Interestinglyspinach IspG another plant MEP pathway enzyme found inchloroplast was reported to accept electrons from photosyn-thetic light reaction [29] and the expression of GbIspH1 wasenhanced by light triggering [18] With all of these reports itis likely that photosynthetic regulation is operating in plantMEP pathway During the photosynthetic light reactionproton is transported from chloroplast stroma to thylakoidlumen and the pH value of stroma approaches pH 80[30] From our pH-dependent activity study it is plausiblethat the N-terminal polypeptide region of GbIspH1 couldmodulate the enzyme activity in a pH-dependent manner inchloroplast

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This work was supported under the framework of Interna-tional Cooperation Program managed by National ResearchFoundation of Korea (NRF-2013K2A1B7A01044254) andby Basic Science Research Program through the NationalResearch Foundation of Korea (NRF) funded by theMinistryof Education (NRF-2010-0020984) The authors also thankto Dr Sun Hee Kim at KBSI for the preliminary EPRmeasurements

References

[1] M Rohmer M Knani P Simonin B Sutter and H SahmldquoIsoprenoid biosynthesis in bacteria a novel pathway for theearly steps leading to isopentenyl diphosphaterdquo BiochemicalJournal vol 295 no 2 pp 517ndash524 1993

[2] T Kuzuyama and H Seto ldquoTwo distinct pathways for essentialmetabolic precursors for isoprenoid biosynthesisrdquo Proceedingsof the Japan Academy Series B Physical and Biological Sciencesvol 88 no 3 pp 41ndash52 2012

[3] W Eisenreich A Bacher D Arigoni and F Rohdich ldquoBiosyn-thesis of isoprenoids via the non-mevalonate pathwayrdquo Cellularand Molecular Life Sciences vol 61 no 12 pp 1401ndash1426 2004

[4] P Adam S Hecht W Eisenreich et al ldquoBiosynthesis of ter-penes studies on 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphos-phate reductaserdquo Proceedings of the National Academy of Sci-ences of the United States of America vol 99 no 19 pp 12108ndash12113 2002

[5] F Rohdich S Hecht A Bacher and W Eisenreich ldquoDeoxyxy-lulose phosphate pathway of isoprenoid biosynthesis Discoveryand function of ispDEFGH genes and their cognate enzymesrdquoPure and Applied Chemistry vol 75 no 2-3 pp 393ndash405 2003

[6] M Seemann K Janthawornpong J Schweizer et al ldquoIso-prenoid biosynthesis via the MEP pathway in vivo Mossbauer

spectroscopy identifies a [4Fe-4S]2+ center with unusual coor-dination sphere in the LytB proteinrdquo Journal of the AmericanChemical Society vol 131 no 37 pp 13184ndash13185 2009

[7] T Grawert F Rohdich L Span et al ldquoStructure of active IspHenzyme fromescherichia coli providesmechanistic insights intosubstrate reductionrdquoAngewandte Chemie International Editionvol 48 no 31 pp 5756ndash5759 2009

[8] T Grawert I Span W Eisenreich et al ldquoProbing the reactionmechanism of IspH protein by X-ray structure analysisrdquo Pro-ceedings of the National Academy of Sciences of the United Statesof America vol 107 no 3 pp 1077ndash1081 2010

[9] I Rekittke J Wiesner R Rohrich et al ldquoStructure of (E)-4-hydroxy-3-methyl-but-2-enyl diphosphate reductase the ter-minal enzyme of the non-mevalonate pathwayrdquo Journal of theAmerican Chemical Society vol 130 no 51 pp 17206ndash172072008

[10] F Rohdich F Zepeck P Adam et al ldquoThe deoxyxylulosephosphate pathway of isoprenoid biosynthesis studies on themechanisms of the reactions catalyzed by IspG and IspHproteinrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 100 no 4 pp 1586ndash1591 2003

[11] Y Xiao Z K Zhao and P Liu ldquoMechanistic studies of IspHin the deoxyxylulose phosphate pathway heterolytic C-O bondcleavage at C4 positionrdquo Journal of the American ChemicalSociety vol 130 no 7 pp 2164ndash2165 2008

[12] W Wang K Wang Y-L Liu et al ldquoBioorganometallic mech-anism of action and inhibition of IspHrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 107 no 10 pp 4522ndash4527 2010

[13] I Span K Wang W Wang et al ldquoDiscovery of acetylenehydratase activity of the iron-sulphur protein IspHrdquo NatureCommunications vol 3 article 1042 2012

[14] S Hecht S Amslinger J Jauch et al ldquoStudies on the non-mevalonate isoprenoid biosynthetic pathway Simple methodsfor preparation of isotope-labeled (E)-1-hydroxy-2-methylbut-2-enyl 4-diphosphaterdquo Tetrahedron Letters vol 43 no 49 pp8929ndash8933 2002

[15] M J A Bailey and J W Noble ldquoQuantitation of proteinrdquoMethods in Enzymology vol 463 pp 73ndash95 2009

[16] M C Kennedy T A Kent M Emptage H Merkle H Beinertand E Munck ldquoEvidence for the formation of a linear [3Fe-4S] cluster in partially unfolded aconitaserdquo Journal of BiologicalChemistry vol 259 no 23 pp 14463ndash14471 1984

[17] F Tan Y Zhang J Wang J Wei Y Cai and X Qian ldquoAnefficient method for dephosphorylation of phosphopeptides bycerium oxiderdquo Journal of Mass Spectrometry vol 43 no 5 pp628ndash632 2008

[18] S-M Kim T Kuzuyama A Kobayashi T Sando Y-JChang and S-U Kim ldquo1-Hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase (IDS) is encoded by multicopy genes ingymnosperms Ginkgo biloba and Pinus taedardquo Planta vol 227no 2 pp 287ndash298 2008

[19] W Xu N S Lees D Hall D Welideniya B M Hoffman andE C Duin ldquoA closer look at the spectroscopic properties ofpossible reaction intermediates in wild-type and mutant (E)-4-hydroxy-3-methylbut-2-enyl diphosphate reductaserdquo Biochem-istry vol 51 no 24 pp 4835ndash4849 2012

[20] H S Kim B-K Shin and J Han ldquoHigh-throughput HDRinhibitor screeningrdquo Journal of the Korean Society for AppliedBiological Chemistry vol 57 no 1 pp 67ndash72 2014

[21] Y Xiao L Chu Y Sanakis and P Liu ldquoRevisiting the IspHcatalytic system in the deoxyxylulose phosphate pathway

8 Bioinorganic Chemistry and Applications

achieving high activityrdquo Journal of the American ChemicalSociety vol 131 no 29 pp 9931ndash9933 2009

[22] B Altincicek E C Duin A Reichenberg et al ldquoLytB proteincatalyzes the terminal step of the 2-C-methyl-D-erythritol-4-phosphate pathway of isoprenoid biosynthesisrdquo FEBS Lettersvol 532 no 3 pp 437ndash440 2002

[23] R C Rohrich N Englert K Troschke et al ldquoReconstitutionof an apicoplast-localised electron transfer pathway involved inthe isoprenoid biosynthesis of Plasmodium falciparumrdquo FEBSLetters vol 579 no 28 pp 6433ndash6438 2005

[24] M Rodrıguez-Concepcion ldquoEarly steps in isoprenoid biosyn-thesis multilevel regulation of the supply of common precur-sors in plant cellsrdquo Phytochemistry Reviews vol 5 no 1 pp 1ndash152006

[25] J A Bick and B M Lange ldquoMetabolic cross talk betweencytosolic and plastidial pathways of isoprenoid biosynthesisunidirectional transport of intermediates across the chloroplastenvelope membranerdquo Archives of Biochemistry and Biophysicsvol 415 no 2 pp 146ndash154 2003

[26] P Pulido C Perello and M Rodrıguez-Concepcion ldquoNewinsights into plant isoprenoidmetabolismrdquoMolecular Plant vol5 no 5 pp 964ndash967 2012

[27] T Grawert J Kaiser F Zepeck et al ldquoIspH protein ofEscherichia coli studies on iron-sulfur cluster implementationand catalysisrdquo Journal of theAmericanChemical Society vol 126no 40 pp 12847ndash12855 2004

[28] M Kwon B-K Shin J Lee J Han and S-U Kim ldquoCharacter-ization of Burkholderia glumae BGR1 4-hydroxy-3-methylbut-2-enyl diphosphate reductase (HDR) the terminal enzyme in2-C-methyl-d-erythritol 4-phosphate (MEP) pathwayrdquo Journalof the Korean Society for Applied Biological Chemistry vol 56no 1 pp 35ndash40 2013

[29] M Seemann B Tse Sum Bui M Wolff M Miginiac-Maslowand M Rohmer ldquoIsoprenoid biosynthesis in plant chloroplastsvia the MEP pathway direct thylakoidferredoxin-dependentphotoreduction of GcpEIspGrdquo FEBS Letters vol 580 no 6 pp1547ndash1552 2006

[30] M Hauser H Eichelmann V Oja U Heber and A LaiskldquoStimulation by light of rapid pH regulation in the chloroplaststroma in vivo as indicated by CO

2solubilization in leavesrdquo

Plant Physiology vol 108 no 3 pp 1059ndash1066 1995

6 Bioinorganic Chemistry and Applications

OH

OPP

OPP

OPP

HMBPP

IPP

DMAPP

OPP

OP

O

O

O

H

P

O

Fe

O

OPP

HFe

ab

a

b

Allylic anion intermediate

H2O

H2O

eminusH+ eminus

minus

Fe4S4Fe4S4

minusO

Ominus

∙

2eminusH+

Figure 5 Proposed IspH-catalyzed reaction mechanism of isopentenyl diphosphate biosynthesis Steps a and b represent possible protontransfer pathways to the allylic anion intermediate which results in different product formation

conformational change of MoFe protein required to separatedocked Fe protein after electron transfer is the rate-limitingstep of nitrogenase catalysisTherefore it is very likely that thereaction step involving separation of the biological electrondonor from IspH would be rate-limiting step Based on theactivation energy comparison it is also proposed that the N-terminal region of GbIspH1 does not actively participate inthe electron transfer step

33 Reaction Products Identification In plant cell bothMVApathway and MEP pathway are found in cytosol and chloro-plast respectively and believed to have different physiologicalfunctions [24] It is also known that MVA pathway producesonly IPP butMEP pathway provides IPP andDMAPP Differ-ent ratio of IPPDMAPP is required for the specific isoprenyldiphosphate biosynthesis in cytosol and chloroplast andapparentlymore IPP thanDMAPP is required for the synthe-sis of geranyl diphosphate (GPP) farnesyl diphosphate (FPP)and so on Even though short chain prenyl diphosphateincluding IPP (C5) DMAPP (C5) GPP (C10) and FPP (C15)can be transported to other cell organelles in plant cell [25]isoprenyl diphosphate isomerase (IDI) is universally foundfrom the cell organelles including chloroplast mitochondriaand peroxisome Hence maintaining the optimummetabolicflux of IPPDMAPP in the cell looks critical [26] Thisbackground information motivated us to study the productsratio of IPPDMAPP produced by IspH

Interestingly the ratio of IPPDMAPP was also recog-nized as an important trait related with the reaction mech-anism According to the proposed IspH-catalyzed reactionmechanisms the allylic anion species is commonly formedby two different IspH reaction mechanism either from thereduction of substrate allylic radical species [7] or from theorganometallic complex formation between substrate andthe Fe

4S4cluster [8] (Figure 5) The allylic anion species

was suggested as the key intermediate by both mechanismsthat controls product formation and therefore determinesthe products ratio Proton transfer from the intermediate

diphosphate group to the allylic anion moiety is the keyreaction step to control the product distribution of IPPand DMAPP So far all IspH proteins including P falci-parum IspH were reported to produce 45ndash63 1 ratio ofIPPDMAPP [27] Recently we have reported that the IDI-missing plant pathogenic bacterium Burkholderia glumaeproduced 2 1 ratio of IPP and DMAPP [28]

Identification of IPP and DMAPP produced by GbIspH1-full and GbIspH1-truncated was achieved by GCMS anal-ysis of the dephosphorylated products of IPP and DMAPP(Figure S5) and the products ratio was obtained by GC-FIDquantitation (Supplementary Material) Controlled experi-ment with mixtures of standard IPP and DMAPP foundthat the recovery efficiency of prenyl and isoprenyl alcoholswas 1 092 and the experimental results were correctedaccordingly Surprisingly the ratios of IPPDMAPP from thereaction mixtures of GbIspH1-full and GbIspH1-truncatedwere both found as 15 1 reproducibly Because the productsratios obtained from GbIspH1 proteins were significantlylarger than the reported values from other IspH proteins itwas proposed that the proton transfer to the allylic anionintermediate during the GbIspH1 catalysis could be differentfrom the bacterial IspH system

4 Conclusions

In summary catalytically active GbIspH1 proteins ofGbIspH1-full and GbIspH1-truncated were successfullypurified and characterized under anaerobic conditions Theisolated GbIspH1 proteins contained the Fe

4S4cluster like

bacterial IspH and it showed comparable catalytic activityto the other IspH proteins The extra N-terminal region ofGbIspH1 common to plant IspH proteins appears not toinfluence the rate-limiting step because GbIspH1-full andGbIspH1-truncated showed similar kinetic parameters of119896cat and activation energy However the extra N-terminalregion was suggested to modulate the enzyme activity in

Bioinorganic Chemistry and Applications 7

a pH-dependent manner based on the pH-dependentactivity profiles of GbIspH1-full and GbIspH1-truncated

GbIspH1-full with complete GbIspH1 polypeptide wasmeasured to have a maximum enzyme activity at pH 80 andIspH from A aeolicus was reported to show the maximumenzyme activity in the range of pH 70ndash75 [22] Interestinglyspinach IspG another plant MEP pathway enzyme found inchloroplast was reported to accept electrons from photosyn-thetic light reaction [29] and the expression of GbIspH1 wasenhanced by light triggering [18] With all of these reports itis likely that photosynthetic regulation is operating in plantMEP pathway During the photosynthetic light reactionproton is transported from chloroplast stroma to thylakoidlumen and the pH value of stroma approaches pH 80[30] From our pH-dependent activity study it is plausiblethat the N-terminal polypeptide region of GbIspH1 couldmodulate the enzyme activity in a pH-dependent manner inchloroplast

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This work was supported under the framework of Interna-tional Cooperation Program managed by National ResearchFoundation of Korea (NRF-2013K2A1B7A01044254) andby Basic Science Research Program through the NationalResearch Foundation of Korea (NRF) funded by theMinistryof Education (NRF-2010-0020984) The authors also thankto Dr Sun Hee Kim at KBSI for the preliminary EPRmeasurements

References

[1] M Rohmer M Knani P Simonin B Sutter and H SahmldquoIsoprenoid biosynthesis in bacteria a novel pathway for theearly steps leading to isopentenyl diphosphaterdquo BiochemicalJournal vol 295 no 2 pp 517ndash524 1993

[2] T Kuzuyama and H Seto ldquoTwo distinct pathways for essentialmetabolic precursors for isoprenoid biosynthesisrdquo Proceedingsof the Japan Academy Series B Physical and Biological Sciencesvol 88 no 3 pp 41ndash52 2012

[3] W Eisenreich A Bacher D Arigoni and F Rohdich ldquoBiosyn-thesis of isoprenoids via the non-mevalonate pathwayrdquo Cellularand Molecular Life Sciences vol 61 no 12 pp 1401ndash1426 2004

[4] P Adam S Hecht W Eisenreich et al ldquoBiosynthesis of ter-penes studies on 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphos-phate reductaserdquo Proceedings of the National Academy of Sci-ences of the United States of America vol 99 no 19 pp 12108ndash12113 2002

[5] F Rohdich S Hecht A Bacher and W Eisenreich ldquoDeoxyxy-lulose phosphate pathway of isoprenoid biosynthesis Discoveryand function of ispDEFGH genes and their cognate enzymesrdquoPure and Applied Chemistry vol 75 no 2-3 pp 393ndash405 2003

[6] M Seemann K Janthawornpong J Schweizer et al ldquoIso-prenoid biosynthesis via the MEP pathway in vivo Mossbauer

spectroscopy identifies a [4Fe-4S]2+ center with unusual coor-dination sphere in the LytB proteinrdquo Journal of the AmericanChemical Society vol 131 no 37 pp 13184ndash13185 2009

[7] T Grawert F Rohdich L Span et al ldquoStructure of active IspHenzyme fromescherichia coli providesmechanistic insights intosubstrate reductionrdquoAngewandte Chemie International Editionvol 48 no 31 pp 5756ndash5759 2009

[8] T Grawert I Span W Eisenreich et al ldquoProbing the reactionmechanism of IspH protein by X-ray structure analysisrdquo Pro-ceedings of the National Academy of Sciences of the United Statesof America vol 107 no 3 pp 1077ndash1081 2010

[9] I Rekittke J Wiesner R Rohrich et al ldquoStructure of (E)-4-hydroxy-3-methyl-but-2-enyl diphosphate reductase the ter-minal enzyme of the non-mevalonate pathwayrdquo Journal of theAmerican Chemical Society vol 130 no 51 pp 17206ndash172072008

[10] F Rohdich F Zepeck P Adam et al ldquoThe deoxyxylulosephosphate pathway of isoprenoid biosynthesis studies on themechanisms of the reactions catalyzed by IspG and IspHproteinrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 100 no 4 pp 1586ndash1591 2003

[11] Y Xiao Z K Zhao and P Liu ldquoMechanistic studies of IspHin the deoxyxylulose phosphate pathway heterolytic C-O bondcleavage at C4 positionrdquo Journal of the American ChemicalSociety vol 130 no 7 pp 2164ndash2165 2008

[12] W Wang K Wang Y-L Liu et al ldquoBioorganometallic mech-anism of action and inhibition of IspHrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 107 no 10 pp 4522ndash4527 2010

[13] I Span K Wang W Wang et al ldquoDiscovery of acetylenehydratase activity of the iron-sulphur protein IspHrdquo NatureCommunications vol 3 article 1042 2012

[14] S Hecht S Amslinger J Jauch et al ldquoStudies on the non-mevalonate isoprenoid biosynthetic pathway Simple methodsfor preparation of isotope-labeled (E)-1-hydroxy-2-methylbut-2-enyl 4-diphosphaterdquo Tetrahedron Letters vol 43 no 49 pp8929ndash8933 2002

[15] M J A Bailey and J W Noble ldquoQuantitation of proteinrdquoMethods in Enzymology vol 463 pp 73ndash95 2009

[16] M C Kennedy T A Kent M Emptage H Merkle H Beinertand E Munck ldquoEvidence for the formation of a linear [3Fe-4S] cluster in partially unfolded aconitaserdquo Journal of BiologicalChemistry vol 259 no 23 pp 14463ndash14471 1984

[17] F Tan Y Zhang J Wang J Wei Y Cai and X Qian ldquoAnefficient method for dephosphorylation of phosphopeptides bycerium oxiderdquo Journal of Mass Spectrometry vol 43 no 5 pp628ndash632 2008

[18] S-M Kim T Kuzuyama A Kobayashi T Sando Y-JChang and S-U Kim ldquo1-Hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase (IDS) is encoded by multicopy genes ingymnosperms Ginkgo biloba and Pinus taedardquo Planta vol 227no 2 pp 287ndash298 2008

[19] W Xu N S Lees D Hall D Welideniya B M Hoffman andE C Duin ldquoA closer look at the spectroscopic properties ofpossible reaction intermediates in wild-type and mutant (E)-4-hydroxy-3-methylbut-2-enyl diphosphate reductaserdquo Biochem-istry vol 51 no 24 pp 4835ndash4849 2012

[20] H S Kim B-K Shin and J Han ldquoHigh-throughput HDRinhibitor screeningrdquo Journal of the Korean Society for AppliedBiological Chemistry vol 57 no 1 pp 67ndash72 2014

[21] Y Xiao L Chu Y Sanakis and P Liu ldquoRevisiting the IspHcatalytic system in the deoxyxylulose phosphate pathway

8 Bioinorganic Chemistry and Applications

achieving high activityrdquo Journal of the American ChemicalSociety vol 131 no 29 pp 9931ndash9933 2009

[22] B Altincicek E C Duin A Reichenberg et al ldquoLytB proteincatalyzes the terminal step of the 2-C-methyl-D-erythritol-4-phosphate pathway of isoprenoid biosynthesisrdquo FEBS Lettersvol 532 no 3 pp 437ndash440 2002

[23] R C Rohrich N Englert K Troschke et al ldquoReconstitutionof an apicoplast-localised electron transfer pathway involved inthe isoprenoid biosynthesis of Plasmodium falciparumrdquo FEBSLetters vol 579 no 28 pp 6433ndash6438 2005

[24] M Rodrıguez-Concepcion ldquoEarly steps in isoprenoid biosyn-thesis multilevel regulation of the supply of common precur-sors in plant cellsrdquo Phytochemistry Reviews vol 5 no 1 pp 1ndash152006

[25] J A Bick and B M Lange ldquoMetabolic cross talk betweencytosolic and plastidial pathways of isoprenoid biosynthesisunidirectional transport of intermediates across the chloroplastenvelope membranerdquo Archives of Biochemistry and Biophysicsvol 415 no 2 pp 146ndash154 2003

[26] P Pulido C Perello and M Rodrıguez-Concepcion ldquoNewinsights into plant isoprenoidmetabolismrdquoMolecular Plant vol5 no 5 pp 964ndash967 2012

[27] T Grawert J Kaiser F Zepeck et al ldquoIspH protein ofEscherichia coli studies on iron-sulfur cluster implementationand catalysisrdquo Journal of theAmericanChemical Society vol 126no 40 pp 12847ndash12855 2004

[28] M Kwon B-K Shin J Lee J Han and S-U Kim ldquoCharacter-ization of Burkholderia glumae BGR1 4-hydroxy-3-methylbut-2-enyl diphosphate reductase (HDR) the terminal enzyme in2-C-methyl-d-erythritol 4-phosphate (MEP) pathwayrdquo Journalof the Korean Society for Applied Biological Chemistry vol 56no 1 pp 35ndash40 2013

[29] M Seemann B Tse Sum Bui M Wolff M Miginiac-Maslowand M Rohmer ldquoIsoprenoid biosynthesis in plant chloroplastsvia the MEP pathway direct thylakoidferredoxin-dependentphotoreduction of GcpEIspGrdquo FEBS Letters vol 580 no 6 pp1547ndash1552 2006

[30] M Hauser H Eichelmann V Oja U Heber and A LaiskldquoStimulation by light of rapid pH regulation in the chloroplaststroma in vivo as indicated by CO

2solubilization in leavesrdquo

Plant Physiology vol 108 no 3 pp 1059ndash1066 1995

Bioinorganic Chemistry and Applications 7

a pH-dependent manner based on the pH-dependentactivity profiles of GbIspH1-full and GbIspH1-truncated

GbIspH1-full with complete GbIspH1 polypeptide wasmeasured to have a maximum enzyme activity at pH 80 andIspH from A aeolicus was reported to show the maximumenzyme activity in the range of pH 70ndash75 [22] Interestinglyspinach IspG another plant MEP pathway enzyme found inchloroplast was reported to accept electrons from photosyn-thetic light reaction [29] and the expression of GbIspH1 wasenhanced by light triggering [18] With all of these reports itis likely that photosynthetic regulation is operating in plantMEP pathway During the photosynthetic light reactionproton is transported from chloroplast stroma to thylakoidlumen and the pH value of stroma approaches pH 80[30] From our pH-dependent activity study it is plausiblethat the N-terminal polypeptide region of GbIspH1 couldmodulate the enzyme activity in a pH-dependent manner inchloroplast

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This work was supported under the framework of Interna-tional Cooperation Program managed by National ResearchFoundation of Korea (NRF-2013K2A1B7A01044254) andby Basic Science Research Program through the NationalResearch Foundation of Korea (NRF) funded by theMinistryof Education (NRF-2010-0020984) The authors also thankto Dr Sun Hee Kim at KBSI for the preliminary EPRmeasurements

References

[1] M Rohmer M Knani P Simonin B Sutter and H SahmldquoIsoprenoid biosynthesis in bacteria a novel pathway for theearly steps leading to isopentenyl diphosphaterdquo BiochemicalJournal vol 295 no 2 pp 517ndash524 1993

[2] T Kuzuyama and H Seto ldquoTwo distinct pathways for essentialmetabolic precursors for isoprenoid biosynthesisrdquo Proceedingsof the Japan Academy Series B Physical and Biological Sciencesvol 88 no 3 pp 41ndash52 2012

[3] W Eisenreich A Bacher D Arigoni and F Rohdich ldquoBiosyn-thesis of isoprenoids via the non-mevalonate pathwayrdquo Cellularand Molecular Life Sciences vol 61 no 12 pp 1401ndash1426 2004

[4] P Adam S Hecht W Eisenreich et al ldquoBiosynthesis of ter-penes studies on 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphos-phate reductaserdquo Proceedings of the National Academy of Sci-ences of the United States of America vol 99 no 19 pp 12108ndash12113 2002

[5] F Rohdich S Hecht A Bacher and W Eisenreich ldquoDeoxyxy-lulose phosphate pathway of isoprenoid biosynthesis Discoveryand function of ispDEFGH genes and their cognate enzymesrdquoPure and Applied Chemistry vol 75 no 2-3 pp 393ndash405 2003

[6] M Seemann K Janthawornpong J Schweizer et al ldquoIso-prenoid biosynthesis via the MEP pathway in vivo Mossbauer

spectroscopy identifies a [4Fe-4S]2+ center with unusual coor-dination sphere in the LytB proteinrdquo Journal of the AmericanChemical Society vol 131 no 37 pp 13184ndash13185 2009

[7] T Grawert F Rohdich L Span et al ldquoStructure of active IspHenzyme fromescherichia coli providesmechanistic insights intosubstrate reductionrdquoAngewandte Chemie International Editionvol 48 no 31 pp 5756ndash5759 2009

[8] T Grawert I Span W Eisenreich et al ldquoProbing the reactionmechanism of IspH protein by X-ray structure analysisrdquo Pro-ceedings of the National Academy of Sciences of the United Statesof America vol 107 no 3 pp 1077ndash1081 2010

[9] I Rekittke J Wiesner R Rohrich et al ldquoStructure of (E)-4-hydroxy-3-methyl-but-2-enyl diphosphate reductase the ter-minal enzyme of the non-mevalonate pathwayrdquo Journal of theAmerican Chemical Society vol 130 no 51 pp 17206ndash172072008

[10] F Rohdich F Zepeck P Adam et al ldquoThe deoxyxylulosephosphate pathway of isoprenoid biosynthesis studies on themechanisms of the reactions catalyzed by IspG and IspHproteinrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 100 no 4 pp 1586ndash1591 2003

[11] Y Xiao Z K Zhao and P Liu ldquoMechanistic studies of IspHin the deoxyxylulose phosphate pathway heterolytic C-O bondcleavage at C4 positionrdquo Journal of the American ChemicalSociety vol 130 no 7 pp 2164ndash2165 2008

[12] W Wang K Wang Y-L Liu et al ldquoBioorganometallic mech-anism of action and inhibition of IspHrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 107 no 10 pp 4522ndash4527 2010

[13] I Span K Wang W Wang et al ldquoDiscovery of acetylenehydratase activity of the iron-sulphur protein IspHrdquo NatureCommunications vol 3 article 1042 2012

[14] S Hecht S Amslinger J Jauch et al ldquoStudies on the non-mevalonate isoprenoid biosynthetic pathway Simple methodsfor preparation of isotope-labeled (E)-1-hydroxy-2-methylbut-2-enyl 4-diphosphaterdquo Tetrahedron Letters vol 43 no 49 pp8929ndash8933 2002

[15] M J A Bailey and J W Noble ldquoQuantitation of proteinrdquoMethods in Enzymology vol 463 pp 73ndash95 2009

[16] M C Kennedy T A Kent M Emptage H Merkle H Beinertand E Munck ldquoEvidence for the formation of a linear [3Fe-4S] cluster in partially unfolded aconitaserdquo Journal of BiologicalChemistry vol 259 no 23 pp 14463ndash14471 1984

[17] F Tan Y Zhang J Wang J Wei Y Cai and X Qian ldquoAnefficient method for dephosphorylation of phosphopeptides bycerium oxiderdquo Journal of Mass Spectrometry vol 43 no 5 pp628ndash632 2008

[18] S-M Kim T Kuzuyama A Kobayashi T Sando Y-JChang and S-U Kim ldquo1-Hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase (IDS) is encoded by multicopy genes ingymnosperms Ginkgo biloba and Pinus taedardquo Planta vol 227no 2 pp 287ndash298 2008

[19] W Xu N S Lees D Hall D Welideniya B M Hoffman andE C Duin ldquoA closer look at the spectroscopic properties ofpossible reaction intermediates in wild-type and mutant (E)-4-hydroxy-3-methylbut-2-enyl diphosphate reductaserdquo Biochem-istry vol 51 no 24 pp 4835ndash4849 2012

[20] H S Kim B-K Shin and J Han ldquoHigh-throughput HDRinhibitor screeningrdquo Journal of the Korean Society for AppliedBiological Chemistry vol 57 no 1 pp 67ndash72 2014

[21] Y Xiao L Chu Y Sanakis and P Liu ldquoRevisiting the IspHcatalytic system in the deoxyxylulose phosphate pathway

8 Bioinorganic Chemistry and Applications

achieving high activityrdquo Journal of the American ChemicalSociety vol 131 no 29 pp 9931ndash9933 2009

[22] B Altincicek E C Duin A Reichenberg et al ldquoLytB proteincatalyzes the terminal step of the 2-C-methyl-D-erythritol-4-phosphate pathway of isoprenoid biosynthesisrdquo FEBS Lettersvol 532 no 3 pp 437ndash440 2002

[23] R C Rohrich N Englert K Troschke et al ldquoReconstitutionof an apicoplast-localised electron transfer pathway involved inthe isoprenoid biosynthesis of Plasmodium falciparumrdquo FEBSLetters vol 579 no 28 pp 6433ndash6438 2005

[24] M Rodrıguez-Concepcion ldquoEarly steps in isoprenoid biosyn-thesis multilevel regulation of the supply of common precur-sors in plant cellsrdquo Phytochemistry Reviews vol 5 no 1 pp 1ndash152006

[25] J A Bick and B M Lange ldquoMetabolic cross talk betweencytosolic and plastidial pathways of isoprenoid biosynthesisunidirectional transport of intermediates across the chloroplastenvelope membranerdquo Archives of Biochemistry and Biophysicsvol 415 no 2 pp 146ndash154 2003

[26] P Pulido C Perello and M Rodrıguez-Concepcion ldquoNewinsights into plant isoprenoidmetabolismrdquoMolecular Plant vol5 no 5 pp 964ndash967 2012

[27] T Grawert J Kaiser F Zepeck et al ldquoIspH protein ofEscherichia coli studies on iron-sulfur cluster implementationand catalysisrdquo Journal of theAmericanChemical Society vol 126no 40 pp 12847ndash12855 2004

[28] M Kwon B-K Shin J Lee J Han and S-U Kim ldquoCharacter-ization of Burkholderia glumae BGR1 4-hydroxy-3-methylbut-2-enyl diphosphate reductase (HDR) the terminal enzyme in2-C-methyl-d-erythritol 4-phosphate (MEP) pathwayrdquo Journalof the Korean Society for Applied Biological Chemistry vol 56no 1 pp 35ndash40 2013

[29] M Seemann B Tse Sum Bui M Wolff M Miginiac-Maslowand M Rohmer ldquoIsoprenoid biosynthesis in plant chloroplastsvia the MEP pathway direct thylakoidferredoxin-dependentphotoreduction of GcpEIspGrdquo FEBS Letters vol 580 no 6 pp1547ndash1552 2006

[30] M Hauser H Eichelmann V Oja U Heber and A LaiskldquoStimulation by light of rapid pH regulation in the chloroplaststroma in vivo as indicated by CO

2solubilization in leavesrdquo

Plant Physiology vol 108 no 3 pp 1059ndash1066 1995

8 Bioinorganic Chemistry and Applications

achieving high activityrdquo Journal of the American ChemicalSociety vol 131 no 29 pp 9931ndash9933 2009

[22] B Altincicek E C Duin A Reichenberg et al ldquoLytB proteincatalyzes the terminal step of the 2-C-methyl-D-erythritol-4-phosphate pathway of isoprenoid biosynthesisrdquo FEBS Lettersvol 532 no 3 pp 437ndash440 2002

[23] R C Rohrich N Englert K Troschke et al ldquoReconstitutionof an apicoplast-localised electron transfer pathway involved inthe isoprenoid biosynthesis of Plasmodium falciparumrdquo FEBSLetters vol 579 no 28 pp 6433ndash6438 2005

[24] M Rodrıguez-Concepcion ldquoEarly steps in isoprenoid biosyn-thesis multilevel regulation of the supply of common precur-sors in plant cellsrdquo Phytochemistry Reviews vol 5 no 1 pp 1ndash152006

[25] J A Bick and B M Lange ldquoMetabolic cross talk betweencytosolic and plastidial pathways of isoprenoid biosynthesisunidirectional transport of intermediates across the chloroplastenvelope membranerdquo Archives of Biochemistry and Biophysicsvol 415 no 2 pp 146ndash154 2003

[26] P Pulido C Perello and M Rodrıguez-Concepcion ldquoNewinsights into plant isoprenoidmetabolismrdquoMolecular Plant vol5 no 5 pp 964ndash967 2012

[27] T Grawert J Kaiser F Zepeck et al ldquoIspH protein ofEscherichia coli studies on iron-sulfur cluster implementationand catalysisrdquo Journal of theAmericanChemical Society vol 126no 40 pp 12847ndash12855 2004

[28] M Kwon B-K Shin J Lee J Han and S-U Kim ldquoCharacter-ization of Burkholderia glumae BGR1 4-hydroxy-3-methylbut-2-enyl diphosphate reductase (HDR) the terminal enzyme in2-C-methyl-d-erythritol 4-phosphate (MEP) pathwayrdquo Journalof the Korean Society for Applied Biological Chemistry vol 56no 1 pp 35ndash40 2013

[29] M Seemann B Tse Sum Bui M Wolff M Miginiac-Maslowand M Rohmer ldquoIsoprenoid biosynthesis in plant chloroplastsvia the MEP pathway direct thylakoidferredoxin-dependentphotoreduction of GcpEIspGrdquo FEBS Letters vol 580 no 6 pp1547ndash1552 2006

[30] M Hauser H Eichelmann V Oja U Heber and A LaiskldquoStimulation by light of rapid pH regulation in the chloroplaststroma in vivo as indicated by CO

2solubilization in leavesrdquo

Plant Physiology vol 108 no 3 pp 1059ndash1066 1995