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Hindawi Publishing CorporationBioMed Research InternationalVolume 2013 Article ID 458571 13 pageshttpdxdoiorg1011552013458571

Research ArticleStructure-Based Mechanism for Early PLP-Mediated Steps ofRabbit Cytosolic Serine Hydroxymethyltransferase Reaction

Martino L Di Salvo1 J Neel Scarsdale2 Galina Kazanina2 Roberto Contestabile1

Verne Schirch3 and H Tonie Wright3

1 Dipartimento di Scienze Biochimiche Sapienza Universita di Roma 00185 Roma Italy2 Center for the Study of Biological Complexity and Institute for Structural Biology and Drug Discovery RichmondVA 23284-2030 USA

3Department of Biochemistry and Institute of Structural Biology and Drug Discovery Virginia Commonwealth UniversityRichmond VA 23219 USA

Correspondence should be addressed to Martino L Di Salvo martinodisalvouniroma1it

Received 6 June 2013 Accepted 26 June 2013

Academic Editor Alessandro Paiardini

Copyright copy 2013 Martino L Di Salvo et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Serine hydroxymethyltransferase catalyzes the reversible interconversion of L-serine and glycinewith transfer of one-carbon groupsto and from tetrahydrofolate Active site residue Thr254 is known to be involved in the transaldimination reaction a crucial stepin the catalytic mechanism of all pyridoxal 51015840-phosphate- (PLP-) dependent enzymes which determines binding of substrates andrelease of products In order to better understand the role of Thr254 we have expressed characterized and determined the crystalstructures of rabbit cytosolic serine hydroxymethyltransferase T254A and T254C mutant forms in the absence and presence ofsubstrates These mutants accumulate a kinetically stable gem-diamine intermediate and their crystal structures show differencesin the active site with respect to wild type The kinetic and crystallographic data acquired with mutant enzymes permit us to inferthat conversion of gem-diamine to external aldimine is significantly slowed because intermediates are trapped into an anomalousposition by a misorientation of the PLP ring and a new energy barrier hampers the transaldimination reaction This barrier likelyarises from the loss of the stabilizing hydrogen bond between the hydroxymethyl group ofThr254 and the 120576-amino group of activesite Lys257 which stabilizes the external aldimine intermediate in wild type SHMTs

1 Introduction

Serine hydroxymethyltransferase (SHMT EC 2121) isa ubiquitous pyridoxal 51015840-phosphate- (PLP-) dependentenzyme that catalyzes the reversible interconversion of L-serine and glycine coupled to the formation and breakdownof 510-methylenetetrahydrofolate (510-CH

2-H4PteGlu) [1

2] Because of its essential role in one-carbon unitsmetabolism SHMT has been often indicated as a potentialtarget of chemotherapeutic agents [3ndash5] It also catalyzesthe conversion of 510-methenylene- to 5-formyl-H

4PteGlu

[6] the transamination and racemization of D- and L-alanine [7] the retro-aldol cleavage of erythro and threoisomers of both L-threonine and L-120573-phenylserine [8] andthe decarboxylation of aminomalonate [9] PLP-dependent

enzymes exist in complexes that absorbs in the 310 nm to500 nm range as the result of a conjugated 120587-electron systemThese absorption properties have played an important rolein elucidating the mechanisms of PLP addition and catalysissince several intermediates on the reaction pathway haveunique structural and absorbance characteristics [10 11]SHMT is distinctive among the PLP-dependent enzymesin the number of these absorbing complexes that can beobserved and these have been exploited to determinatekinetic rates for their interconversion by stopped-flow andtemperature jump spectroscopy [11ndash13]

In PLP-dependent enzymes the 41015840-aldehyde of PLP isbound as an aldimine to the 120576-amino moiety of an active siteLys residue in what is called the ldquointernal aldiminerdquo For thosePLP-dependent enzymes that catalyze reactions involving

2 BioMed Research International

External aldimine

HHC

H R

NH

HHC

R

H

HR

Internal aldimine

HHC

H R EnzEnz

C

HHC

HR

H

HEnz

H

Enz

N

Hydrated internal aldimine

HN

HC

Enz

HO

Quinonoid intermediate

O

HN

HC

Enz

Internal aldimine

(carbinolamine Amax

asymp 340nm)

H+

2minusO3PO

NH+

2minusO3PO

Ominus

Ominus

Ominus

CH2

NH+

2minusO3POCH2

Lys257

Lys257 Lys257Lys257

Lys257

Lys257

(ketoenamine form Amax

asymp 430nm)

NH+

N+ N+ N+ N+

N+

2minusO3POCH2

COOminus

COOminus COOminus

COOminus

COOminus

NH NH2

(enolimine form Amax

asymp 340nm)

(Amax

asymp 343nm) (Amax

asymp 343nm)(A

maxasymp 426nm)

(Amax

asymp 498nm)

Gem-diamine I

H2 C

HC

NH+

NH+

2minusO3PO 2minusO3PO

2minusO3PO

Ominus

Ominus

Ominus

Gem-diamine II

H2

CH2

CH2

OHminus

N

Scheme 1 Mechanism of transaldimination reaction of SHMT

substrate amino acids the initial step in the catalytic reactionis the formation of the ldquogeminal diaminerdquo (gem-diamine)between the C41015840 aldehyde of PLP and the amino group ofthe substrate The orientation of the substrate in the activesite with respect to the plane of PLP to which it is covalentlylinked through its amino group in turn determines whichof the three substituent bonds on C120572 of the substrate will becleaved In 1966 Dunathan [14] provided a unifying conceptfor the specific selection of the substrate scissile bond in PLP-dependent enzymes which has been confirmed in solutionand structural studies In his proposal all bonds broken andmade on the catalytic pathway are the nearest perpendicularto the conjugated 120587 system of the PLP ringThe resulting car-banion at C120572 of the substrate amino acid is stabilized by reso-nance with the 120587-electron system in the pyridine ring of PLPThis intermediate on the catalytic pathway is referred to as theldquoquinonoidrdquo complex and absorbs near 500 nm (Scheme 1)Solution studies have shown that SHMT passes throughseveral ordered spectrophotometrically identifiable interme-diates that reflect changes in the electron system of the PLPcofactor [12 15] and are consistent with Dunathanrsquos proposal

Prior to the availability of crystallographic structureinformation on SHMT it was noted that the amino acidsequences of SHMTs from diverse species have a conservedrun of 4 threonine residues terminating 2 residues upstreamof the active site lysine V-V-T-T-T254-T-H-K257(PLP)-T(numbering is that for rabbit cytosolic serine hydroxymethyl-transferase (rcSHMT)) [16] To determine the possible rolesof this conserved sequence in SHMTcatalysis each threonineof this active site stretch was mutated in ecSHMT to analanine and the effects of the changes on the spectral andkinetic properties were investigated [17] It was found that

only the T226A mutant of ecSHMT (Thr226 in ecSHMTnumbering is equivalent to rcSHMTThr254) had significantspectral and kinetic differences from the wild type enzymeThere was a 32-fold lower 119896cat in the conversion of L-serine toglycine and the T226A mutant was virtually inactive towardcleavage of L-allo-threonine compared to wild type ecSHMTFurthermore in the presence of L-serine the T226A mutantexhibited a large spectral absorbance peak at 343 nm whichis characteristic of a gem-diamine intermediate and only asmall peak at 425 nm characteristic of the external aldimineStopped-flow analysis showed that the 343 nm peak wasformed rapidly but its conversion to the 425 nm absorbingpeak was slow [17] Since the gem-diamine is generally ashort-lived intermediate on the reaction pathways of PLP-dependent enzymes this T226A ecSHMTmutant offered theopportunity to investigate the structural changes that appar-ently slowed the conversion of the gem-diamine intermediateto the external aldimine We were unable to obtain crystalsfor the T226A mutant of ecSHMT However we were ableto crystallize the homologous T254A and T254C mutants ofrcSHMT We report here the kinetic properties and crystalstructures of the T254 mutants of rcSHMT and their glycineand L-serine complexes In addition we have increased theresolution of the structure of wild type rcSHMT to 21 A as anaid in examining details of the gem-diamine structures

2 Results and Discussion

21 Spectroscopic Studies Wild type rcSHMT exhibits acharacteristic major single absorption band with maximumintensity at 430 nm due to the protonated internal aldiminebetween enzyme and cofactor Also aminor band is observed

BioMed Research International 3

at 340 nm (Scheme 1) The addition of saturating concentra-tions of glycine results in a decrease of the major absorptionband with a slight blue shift at 426 nm and the appearanceof a well-defined band at 343 nm A small band centeredat 498 nm is also visible It is well established that theseabsorption bands correspond to the formation of the externalaldimine the gem-diamine and the quinonoid intermediatesrespectively [1 12 18] All absorption bands of wild type andmutant enzymes are shown in Figure 1

Like the wild type the T254A mutant form exhibitsa 430 nm absorption band (although much less intensethan wild type) indicative of the presence of an internalaldimine and also shows an important band at around340 nm that may represent either the enolimine form or thecarbinolamine a hydrated form of the internal aldimine inwhich the water molecule mimics the substrate nucleophilicattack and the gem-diamine formation As shown in Figure 1this band is usually present in very low concentration inthe freshly purified wild type enzyme The addition ofa saturating concentration of glycine to T254A mutantresults in the almost complete loss of absorbance at 430 nmwith a concomitant increase of absorbance at 343 nm Thiseffect was also observed with E coli SHMT T226A mutant(residue Thr226 of ecSHMT corresponds to residue Thr254in rcSHMT) Kinetic studies on the latter mutant showed thatthe complex absorbing at 343 nm is formed in a bimolecularstep providing strong evidence that it is indeed the gem-diamine intermediate [17] The addition of H

4PteGlu to

glycine-saturated wild type SHMT results in the increase ofthe 498 nm absorbing band corresponding to the quinonoidintermediateThe large increase in absorbance below 400 nmis the absorbance of the excess H

4PteGlu Instead when

the T254A mutant was saturated with glycine even in thepresence of H

4PteGlu it did not show any 498 nm absorbing

bandThe effects of the addition of a saturating concentrationof L-serine to T254A rcSHMT mutant enzyme are similarto those observed for glycine with a decrease of the 430 nmabsorbing band and a concomitant increase of the 343 nmabsorbing species In contrast when wild type rcSHMT issaturated with L-serine there are a marked increase anda blue shift of the major absorbing band centered now at426 nm but no absorbance is observed at around 340

The spectral features of the unliganded T224Cmutant arelargely similar to those of the wild type enzyme Howeverthe 340 nm band is slightly more intense If compared tothe T254A mutant the relative intensities of the 430 nm and340 nm bands are reversed Spectral changes upon substratesaddition are similar to the ones observed with the T254Amutant except that a residual absorbance is shown in the420ndash430 nm region

22 Kinetics Studies The spectral properties of the rcSHMTT254A and T254C mutant enzymes described above sug-gest that the gem-diamine intermediate accumulates uponsubstrate addition The purified mutant enzymes were testedfor catalytic activity using L-serine and L-allo-threonineas substrates (Table 1) For L-serine both mutant enzymesshowed slightly increased 119870

119898values (about 2-fold) when

compared to wild type rcSHMT 119896cat values decreased 46-fold

for the Thr to Ala mutant and 531-fold for the Thr to Cysmutant Similarly119870

119898values for L-allo-threonine were found

to be only slightly higher than wild type (less than 2-fold forboth mutants) whereas 119896cat values showed a 9- to 29-folddecrease for T254A and T254C mutants respectively

To better understand the role of Thr254 in the formationand breakdown of the gem-diamine intermediate and thuson the transaldimination reaction the rate of the spectralchanges occurring when L-serine and glycine were added tothe enzyme was determined at different pH values by meansof stopped-flow measurements The 119896on and 119896off values forthe rate of formation and breakdown of the enzyme-substratecomplex absorbing at 343 nm were determined from a plotof 119896obs versus substrate concentration after linear regressionof data and extrapolation of the slope and intercept values(Table 2) For bothmutants and for both substrates at each pHvalue 119896obs was a linear function of substrate concentration asexpected for a second-order reaction The second-order rateconstants for substrate addition and gem-diamine formation(119896on) were corrected for the concentration of the amino acidanionic form at each pH value and are listed as 1198961015840on Theanionic form of the 120572-amino group is assumed to be thetrue substrate for the transaldimination reaction In the pHrange used in the experiments (64ndash80) the concentrationof the anionic form of the amino acid substrates increasesabout 40 times The 119896on values increased with pH and variedfrom 33 sdot 104 to 107 sdot 104 sminus1sdotMminus1 when L-serine was usedas substrate and from 08 sdot 104 to about 44 sdot 104 sminus1sdotMminus1when glycine was used as substrate 119896on values were verysimilar for both T254 mutant forms After correction forthe concentration of anionic substrate 1198961015840on values showed a10-fold decrease as pH increased from 64 to 80 and rangedfrom 20 sdot 106 to 18 sdot 106 sminus1sdotMminus1 with L-serine and from13 sdot 10

6 to 18 sdot 106 sminus1sdotMminus1 with glycine The sigmoidaldependence of 1198961015840on on pH suggests the titration of a groupat the active site involved in a general acid catalysis witha pK119886around 68 (Figure 2) If the internal aldimine of the

mutant enzymes is mostly in the carbinolamine form asinferred from the absorption spectra shown in Figure 1 itmust be dehydrated (through the protonation of the hydroxylgroup followed by the elimination of water) in order to beconverted into the ketoenamine that is able to react with theincoming amino group of the substrates Importantly wehave observed that the ratio of the 428 nm and 340 nm bandschanges with pH in a similar manner with the ketoenamineform being favored at higher pH values (data not shown) Apossible candidate for this acid catalysis is Tyr73 which isinvolved in a cation-120587 interaction with Arg263 It has beenshown that Tyr residues involved in cation-120587 interactionsmay have their pK

119886lowered by 1 to 3 pH units (the normal

pK119886of tyrosine residue is about 95) [19] Furthermore

this residue points toward the protein region where thetransaldimination reaction takes place and its phenolichydroxyl is located in close proximity to C41015840 (see Figure 6)When L-serine was used as substrate 119896off values showed adecreasing trend as pH was increased and ranged from 103to 31 sminus1 for the T254Amutant form and from 39 to 23 sminus1 forT254C In contrast when glycine was used as substrate 119896off


000

004

008

012

016

Wavelength (nm)300 350 400 450 500

Wild type

+gly

+gly

No ligand

Abso

rban

ce

+H4PteGlu

(a)

Wavelength (nm)300 350 400 450 500 550

Wild type

+L-ser

No ligand

000

004

008

012

016

Abso

rban

ce

(b)

000

002

004

006

008

Wavelength (nm)300 350 400 450 500

T254A

+gly

+gly

No ligand

Abso

rban

ce

+H4PteGlu

(c)

Wavelength (nm)300 350 400 450 500 550

T254A

+L-ser

No ligand

000

002

004

006

008

Abso

rban

ce

(d)

Wavelength (nm)300 350 400 450 500

000

002

004

006

008T254C

+gly

No ligand

Abso

rban

ce

+gly+H4PteGlu

(e)

Wavelength (nm)300 350 400 450 500 550

T254C

+L-ser

No ligand

000

002

004

006

008

Abso

rban

ce

(f)

Figure 1 Absorption spectra of wild type and mutant rcSHMT in the absence and presence of substrates Left panels show the absorptionspectra of wild type T254A and T254C rcSHMTs at 30∘C before (black lines) and after the addition of 90 saturating glycine (blue lines)Green lines are spectra taken after the addition of 100 120583M H

4PteGlu to the samples containing glycine Right panels show the absorption

spectra of the same enzymes before (black lines) and after the addition of 90 saturating L-serine (red lines)

values slightly increased with pH ranging from 1 to 29 sminus1for the T254Amutant form and from 51 to 70 sminus1 for T254C

Stopped-flow studies done many years ago at pH 73 forwild type rcSHMT [12] gave a comparable 119896on value to thevalues for the T254A and T254Cmutants On the other hand

the 119896off values determinedwith themutant enzymes are about2 orders of magnitude lower than the wild type These datasuggest that the mutations have not significantly affected therate of formation of the gem-diamine intermediate but have amore significant effect on the rate of its breakdown


0

5

10

15

20

25

60 65 70 75 80 85 90pH

T254A

k998400 on

(sminus1

Mminus1times106)

(a)

60 65 70 75 80 85 900

5

10

15

20

pH

T254C

k998400 on

(sminus1

Mminus1times106)

(b)

Figure 2Dependence of 1198961015840on values determined from stopped-flow experiments on pH for T254A andT254C rcSHMTmutants Open circlescorrespond to data obtained with L-serine while closed circles correspond to data obtained with glycine The continuous lines through theexperimental points were obtained through a least square minimization process using the equation for a sigmoidal curve (the software usedwas GraphPad Prism)

Table 1 Kinetic constants for the hydroxymethyltransferase andretro-aldol cleavage reactions catalyzed by wild type and T254mutant rcSHMT forms

Substrate Kineticconstant

Form of rabbit cytosolic SHMTWild type T253A T253C

L-Serine 119870119898(mM) 03 06 05119896cat (minminus1) 850 185 16

L-allo-Threonine 119870119898 (mM) 15 25 26119896cat (minminus1) 130 143 45

If the rapid spectroscopic changes occurring when theenzyme was mixed with saturating concentration of sub-strates were followed for a longer period of time (up to 50seconds) a slow first-order increase was observed at 426 nmFor the T254A mutant with saturating concentration of L-serine the rate constant was determined to be 10minminus1 Thisvalue is in agreement with previous studies on the T226Amutant of ecSHMT [17] It is interesting to notice that thisrate constant is very close to the 119896cat measured for bothhydroxymethyltransferase and retro-aldol cleavage reactionscatalyzed by this mutant This shows that for the T254Amutant the conversion of the gem-diamine into the externalaldimine intermediate has become the rate-limiting step

The spectral and kinetic studies both strongly suggest thatremoving the hydroxymethyl group of Thr254 by replacingit with either an Ala or a Cys residue greatly slows theconversion of the gem-diamine intermediates to the externalaldimine This may be the result of either blocking theconversion of gem-diamine I to gem-diamine II or of slowingconversion of gem-diamine II to the external aldimineThere-fore the slowing of the catalytic cycle could be explained byeither the mutant gem-diamine complexes being in a morestable form (in an energy well) compared to the wild typeenzyme or by an increased energy barrier to pass to the

external aldimine These two hypotheses are not mutuallyexclusive and could be applied at the same time

23 Crystal Structures We determined the crystal structuresof the T254A and T254C mutant enzymes and the complexof each with glycine and L-serine and the structure of wildtype rcSHMT to a higher resolution than had previouslybeen attained [20] Table 3 lists the crystallographic andrefinement data for the new rabbit cytosolic SHMT structuresdetermined and described in this work All structures exceptfor the T254C-glycine complex were solved in space groupP41with a tetramer of 4 independently determined subunits

per asymmetric unitThe T254C-glycine complex was solvedin space group P4

1212 with a dimer per asymmetric unitThe

oligomeric structure of rcSHMT consists of two tight dimerseach made of identical monomers These tight dimers areloosely associated to form a so-called dimer of dimers Disor-dered density recurred in the insert segment around residue272 and at the amino termini of all monomer subunits asobserved in previously determined SHMT structures butthese disordered residues are not close to the regions of theenzyme around the PLP and substrate binding site which arethe subject of this study

231 Differences in Structure of Unliganded Wild Type andT254 Mutants Except for the small differences in the activesite described herein all structures are virtually identical(rmsd lt04 A in all cases) The interactions of the PLPcofactor with the protein in the wild type and T254 mutantsare almost identical The distance of the Asp228 carboxylateto N1 of the pyridine ring is unchanged in the mutants as arethe noncovalent bond constraints on the phosphoryl groupof the PLP tail and the coplanarity of the His148 imidazolewith the PLP ring The active sites of all subunits of thewild type have phosphate or MES buffer anions bound wherethe carboxylate moiety of the amino acid substrate binds


Table 2 Kinetic constants for the rate of formation and breakdown of the gem-diamine complex for mutant rcSHMT forms with glycine andL-serine substrates

Substrate pH T253A T253C119896on (s

minus1 Mminus1 sdot 104) 1198961015840

on (sminus1 Mminus1 sdot 106) 119896off (s

minus1) 119896on (sminus1 Mminus1 sdot 104) 119896

1015840


minus1)

L-Serine

64 33 20 103 29 18 39

68 58 14 83 40 10 36

72 87 86 58 68 67 32

76 95 37 53 87 34 27

80 107 18 31 94 15 23

Glycine

64 08 13 10 05 85 51

68 13 80 13 07 45 67

72 21 50 19 15 38 69

76 30 29 24 21 22 70

80 44 18 29 37 16 68

in SHMT-substrate complexes (see next paragraph) In theT254A mutant this site is occupied by phosphate ions in onedimer and by MES in the other dimer In the T254C mutantthis anionic site is occupied by water in three subunits and byan apparent phosphate in the fourth subunit (Table 4)

There are significant differences between the wild typeand the T254 mutants in the conformation of the PLPmethylene phosphate tail although these are seen to a differ-ent extent in different subunits (Figure 3) These differencescorrelate with the absence of the C120573 methyl group in theAla254 and Cys254 side chains in the mutants In the wildtype structure this methyl group of Thr254 projects towardthe methylene phosphate tail Replacement of the side chainby Ala or Cys opens a small space that allows the cofactortail to assume a different conformation moving the C51015840towards this cavity This is the most evident change observedin the mutants The fact that not all of the T254A and Csubunits show this change to the same extent suggests thatthere is a low barrier to this switch of conformations andthat the crystallization process is selecting out an asymmetricdistribution As a consequence of the elbow-like rotationof the methylene phosphate PLP tail in the mutant T254forms the PLP ring orientation also diverges from that of thewild type Moreover in the mutant enzyme structures withthe largest PLP tail conformational differences the aldiminelinkage between C41015840 and the 120576-amino group of the active sitelysine (Lys257) is far from being in the plane of the PLP ring(Figure 3(d)) as required by the double bond conjugationpresent in the ketoenamine form of the internal aldimineAs the aldimine linkage is driven away from this plane itsconjugation with the 120587-electron system of the pyrimidinering is diminished and themaximumabsorbance is shifted tolower wavelengths [21] as observed for the mutant enzymesin solution (Figure 1) As discussed above we cannot excludethat the 340 nm absorbing band of the unliganded mutantenzymesmay correspond to the hydrated form of the internalaldimine (carbinolamine Scheme 1) In the internal aldimineform of the T254C mutant the conformational change of themethylene phosphate tail and the displacement of the PLPring are less evident (data not shown)This correlates with theobservation that in the absorption spectrum of this mutant

in solution the amount of the canonical 428 nmketoenamineband is higher than in the T254A mutant

232 The Glycine and L-Serine Complexes of T254A andT254C Mutants The structures of the T254A and T254Cmutants with glycine and L-serine substrate ligands are allin the gem-diamine form (Table 4) showing both the aminogroup of the substrate and the 120576-amino group of Lys257forming bonds to C41015840 of PLP In all subunits of both mutantsand with both substrates the orientation of the PLP ring andthe conformation of the methylene phosphate tail are verysimilar Interestingly the inhomogeneity seen in the internalaldimine structures of the mutant enzymes has disappearedupon binding of substrates For example Figure 4 shows acomparison between the active site structures of the T254Amutant with either L-serine (a) or glycine (b) bound to C41015840of PLP in the gem-diamine form and the internal aldimineThe comparison is made with the subunit of the unligandedmutant enzyme in which the internal aldimine shows thelargest difference with respect to wild type (Figure 3(d))It can be seen that in both gem-diamine structures theorientation of the PLP ring and the conformation of themethylene phosphate tail and of the active site lysine (Lys257)are very similar to those of the internal aldimine form andtherefore differ significantly from the wild type internalaldimine structure The carboxylate group of substrates isoriented to form dual hydrogen bonds with Arg402 andalso with Y83 Residues Ser203 and His231 make hydrogenbonds with the O31015840 of PLP as already observed in all otherSHMT structures In the L-serine gem-diamine complex thehydroxyl group of the substrate makes H-bond interactionswithGlu75 andTyr83 as also observed in the crystal structureof the Bacillus stearothermophilus SHMT-L-serine complex[22]

Wild type rcSHMT enzyme cocrystallized with glycineand 5-CHO-H

4PteGlu showed two of the enzyme subunits in

the gem-diamine form [19] (Table 4) No external aldimineswere present as the other two subunits were in the internalaldimine form It is worth noting that structural variationamong subunits in ligand binding site occupancy and type ofintermediate complex is also found in other eukaryotic and


Table3Cr

ystallo

graphicd

atac

ollectionandrefin

ementstatistic

sfor

rcSH

MTstr

ucturesd

etermined

inthiswork

Wild

type

T254A

T254A+gly

T254A+L-ser

T254C

T254C+gly

T254C+L-ser

Cellspace

grou

p115611561560

P41

115111511574

P41

115011501568

P41

115511551563

P41

114011401547

P41

114611461569

P412

12114

011401556

P41

Resolutio

n119

0ndash210

(216

ndash210

)1118

ndash255(262ndash255)

200ndash265(272ndash265)

115ndash240

(246ndash

240

)1118

ndash240(246ndash

240

)900ndash265(272ndash265)

1118


Com

pleteness()

985(982)

922(879

)996(998

)883(858)

901(869)

696(620)

909(769)

119877work

199(2

48)

201

(306)

203

(292)

197(2

47)

195(2

42)

221

(333)

202

(284)

119877fre

e242

(316)

271

(432)

277

(399)

258

(318)

256

(346

)305

(414)

263

(362)

119873work

105631

(766

8)55295(3849)

52905(3769)

63363(4478)

62364(4393)

19976(1244)

52787(5963)

119873fre

e117

36(845)

6204

(426)

5968

(406)

7156

(510)

7009

(470)

1654

(96)

5963

(375)

⟨119861protein⟩

308

448

34373

394

366

38⟨119861water⟩

398

438

346

373

419

NA

401

RMSD

from

ideal

Bond

leng

ths

0008

0007

0007

000

6000

6000

60007

Bond

angles

1312

412

2113

113

108

116

Ramachand

ranplot

Favored

1460

1424

1403

1425

1435

702

1427

Additio

nally

allowed

123

142

142

146

133

69128

Generou

slyallowed

138

912

156

12Fo

rbidden

37

95

57

10MolProb

ityscore

173(93

)204

(96

)204

(97

)18

8(96

)18

1(97)

224

(93

)200

(96

)


Thr254Ala254

Lys257

(a)

Thr254Ala254

Lys257

(b)

Thr254Ala254

Lys257

(c)

Thr254Ala254

Lys257

(d)

Figure 3 Superposition of the active site structures of rcSHMT wild type (salmon) and T254A (cyan) in the internal aldimine form Panels(a) to (d) correspond to different subunits of the tetramer

prokaryotic SHMTs [22ndash26] The above wild type rcSHMT-glycine-5-CHO-H

4PteGlu ternary complex structure and

that of mouse cytosolic SHMT (which was also cocrystallizedwith glycine and 5-CHO-H

4PteGlu [25]) are the only wild

type SHMT structures in which gem-diamine intermediatescan be seen

When wild type and T254A rcSHMT-glycine gem-diamine structures are superimposed some striking differ-ences can be noticed (Figure 5) The methylene phosphatetail conformation and the orientation of the PLP ring aresignificantly different The carboxylate group of glycine inwild type rcSHMT gem-diamine although still interactingwith Y83 and Arg402 through one of its oxygen atoms isno more oriented to interact optimally with Arg402 and theother oxygen points away from it Moreover the positionof the two amino groups in the gem-diamine is differentbetween wild type and mutant (see arrows in Figure 5) Inparticular the hydrogen atoms of the amino groups pointaway from Tyr73 which might be involved in the proton

exchange required to interconvert the two gem-diamineintermediates (see below for discussion)

In the wild type gem-diamine the bond between the sub-strate amino group andC41015840 of PLP lies roughly perpendicularto the cofactor ring indicating a strong similarity to gem-diamine I intermediate in which the amino group of thesubstrate has attacked the C41015840 Schiff base of the cofactorwith a trajectory that is perpendicular to the pyridine ring(Scheme 1) It appears that the wild type rcSHMT in thecrystal has been trapped as a gem-diamine I intermediatethat barely accumulates in solution On the other handthe gem-diamine forms found in T254 mutants seem tobe in an aberrant position The best way to appreciate thisis to superimpose the structures of wild type gem-diamine(Figure 6(a)) or T254mutant gem-diamine (Figure 6(b))withwild type internal and external aldimines In SHMTs asPLP reacts with the substrate and the internal aldimine isconverted into the external aldimine the pyridine ring rotatesby about 25∘ primarily around the C2-C5 axis This is also


Ala254

Glu75

Tyr83

Arg402

Ser203

His231

Lys257

(a)

Ala254

Glu75

Tyr83

Arg402

Ser203

His231

Lys257

(b)

Figure 4 Superposition of the active site structures of the T254Amutant in the internal aldimine form (cyan) and as a gem-diamine complex(orange) with either L-serine (a) or glycine (b)

Tyr73 Tyr83

Arg402

Thr254Ala254

Figure 5 Superposition of the active site structures of the wild type(slate) andT254Amutant form (orange) of rcSHMTas gem-diaminecomplexes with glycine (pdb 1ls3) The arrows in the figure pointtowards the amino groups of the gem-diamine complexes

observed in prokaryotic B stearothermophilus SHMT withboth glycine and L-serine as substrates and in the ternarycomplex with glycine and 5-CHO-H

4PteGlu [22] In the wild

type rcSHMT gem-diamine the PLP ring lies between thepositions observed in the internal and external aldiminesStrikingly in the mutant T254 gem-diamine the cofactor ringhas a different orientation which is unlikely to correspondto that occurring in the transaldimination reaction This isobviously a consequence of the mutation since this position

of the PLP ring is also observed in the unliganded formsof T254A and T254C rcSHMT structures The gem-diaminestructures of the mutant enzyme appear to be close to thegem-diamine II intermediate (Scheme 1) and the angle ofthe bond from C41015840 of the PLP ring to the 120576-amino group ofK257 being close to the 90∘ predicted for this intermediateII in which the amino group of Lys257 has to be eliminatedin order to form the external aldimine intermediate More-over the carboxylate group of the substrate makes optimaldual hydrogen bonds with Arg402 (Figure 6(b) structurein orange) as is observed in the external aldimine form ofwild type SHMT-glycine complex (Figure 6(b) structure inmagenta)

These observations suggest that in the T254 rcSHMTcrystals the PLP-substrate complex is blocked in the formof an anomalous and stable gem-diamine intermediate Asmentioned above it is noteworthy that in the T254 mutantgem-diamine intermediates the position and orientation ofthe two amino groups point away from Tyr73 compared tothe wild type gem-diamine structure Tyrosine 73 was shownin E coli and bsSHMT to have an important role in thetransaldimination process and was proposed to act as protonexchanger between gem-diamines I and II Interestingly Tyrto Phe mutants of this residue also accumulate the gem-diamine intermediate [18 27]

An additional remarkable variation in the T254 mutantstructures compared to wild type is observed in the peptidebond between His256 and Lys257 Among the determinedmutant structures this peptide bond adopts two distinctorientations one similar to the wild type rcSHMT andthe other differing by up to 180∘ The local variation inconformation of the His256-Lys257 peptide bond may belinked to the rcSHMT T254 mutations since in the internal


Tyr73 Tyr83

Arg402

Lys257

(a)

Tyr73 Tyr83

Arg402

Lys257

(b)

Figure 6 Superposition of wild type structures corresponding to the internal aldimine (salmon rcSHMT) and external aldimine (magentamcSHMT pdb 1eji) forms of the cofactor with the glycine gem-diamine form of the wild type (slate) and T254A mutant (orange)

aldimine of wild type rcSHMT the carbonyl oxygen ofHis256 which is flipped in some mutant structures makesa hydrogen bond through a water molecule to the Thr254side chain The lack of uniformity in structure among thesubunits of the T254 mutants in the conformation of thePLP methylene phosphate tail the position of the PLP ringand the orientation of the His256-Lys257 peptide bondsuggest that mutation of T254 relaxes some local structuralconstraints around the active site

3 Conclusions

The T254A and T254C mutations have created a smallempty space in the active site of rcSHMT due to theabsence of the threonine methyl group This has allowedthe methylene phosphate tail of PLP to adopt a stableuncharacteristic conformation that is in turn responsible foran aberrant positioning of the PLP ring This clearly affectsthe transaldimination step of the SHMT reaction making itthe rate-limiting step in the catalytic cycle In these mutantswhen either L-serine or glycine is added to the enzyme gem-diamine intermediates greatly accumulate This may be theresult of either blocking the conversion of gem-diamine I togem-diamine II slowing the conversion of gem-diamine II tothe external aldimine or both

Conversion of the gem-diamine II into the externalaldimine requires that the 120576-amino group of Lys257 is proto-nated and that it leaves perpendicularly from the si face of C41015840of PLP In Scheme 1 this is shown as a direct proton transferfrom the substrate amino group in gem-diamine I to theleaving amino group of Lys257 in gem-diamine II Howeverthis proton transfer almost certainly does not occur directlybut through shifts of protons in a network of acid-base groupsat the active site It is possible based on the wild type andT254mutant structures that protonation of the Lys257 amineof the gem-diamine is mediated by Tyr73 The position of thegem-diamine amino groups relative to the Tyr73 side chain

varies in the mutants These local structure changes in T254mutants could be responsible for a perturbation of the protontransfer chain which slows the transaldimination reaction

Available external aldimine SHMT structures (such asE coli SHMT as ternary complex with glycine and 5-CHO-H4PteGlu (pdb 1dfo) and bsSHMT with glycine and L-serine

(pdb 1kl1 and 1kkp resp)) all show a good hydrogen bondbetween the Lys257 120576-amino group and the hydroxyl group oftheThr254 side chain as originally suggested by Pascarella etal [16]Thenucleophilicity of the 120576-amino group and theNH

2

of the substrate must be balanced so that transaldiminationcan occur rapidly from either direction The hydrogen bondbetween Thr254 and the Lys257 120576-amino group is likely tobe a critical determinant of this balance In the T254A andT254C mutants which lack the hydroxyl group this H-bondcannot be formed We suggest that the loss of this hydrogenbond destabilizes the external aldimine of the T254 mutantsrelative to the gem-diamine intermediates and results in thetrapping of the latter

PLP-dependent enzymes typically catalyze the reversibletransaldimination reaction between the internal and externalaldimines very rapidly within the dead time of stopped-flowmeasurements and do not accumulate gem-diamine inter-mediates This is a crucial step in the catalytic mechanism ofall PLP-dependent enzymes because it determines binding ofsubstrates and release of products Our studies show that asingle semiconservativemutation of an active site residue canbe critical for the transaldimination in SHMT

4 Materials and Methods

41 Materials All chemicals coenzymes antibiotics andbuffers were from Sigma-Aldrich (St Louis MO USA)or FisherScientific (Pittsburgh PA USA) (6S)-H

4PteGlu

and (6S)-5-CHO-H4PteGlu were gifts from Merck Eprova

AG (Schaffhausen Switzerland) Crystallization buffers werefrom Hampton Research (Laguna Niguel CA USA)


Table 4 Rabbit cytosolic SHMT structures compared in this studyThe table shows the cofactor form and the ligand present in eachsubunit of the structures

Enzyme Chain Form Ligand

Unliganded wild type

A ia PO42minus

B ia PO42minus

C ia MESD ia MES

Unliganded T254A

A ia PO42minus

B ia PO42minus

C ia PO42minus

D ia PO42minus

T254A + glycine

A gd GlyB gd GlyC gd GlyD gd Gly

T254A + L-serine

A gd L-SerB gd L-SerC gd L-SerD gd L-Ser

Unliganded T254C

A ia H2OB ia H2OC ia H2OD ia PO4

T254C + glycine A gd GlyB gd Gly

T254C + L-serine


Wild type + glycine + 5-CHO-H4PteGlu3

A gd GlyB ia mdashC gd GlyD ia mdash

ia internal aldiminegd gem-diamineMES 2-(N-morpholino)ethanesulfonate5-CHO-H4PteGlu3 triglutamic form of 5-formyltetrahydrofolate

42 Mutagenesis Expression and Purification of SHMTMutants were made using the QuikChange site-directedmutagenesis kit from Stratagene (La Jolla CA USA) onrcSHMT cDNA in the pET22b vector [28] The T254A andT254C mutant forms were produced using the primers51015840-CGTGGTGACCACCGCGACCCACAAGACGC-31015840 and51015840-CGTGGTGACCACCTGCACCCACAAGACGC-31015840respectively and their complementary oligonucleotides(the mutated codons are underlined) Each mutationwas confirmed by sequencing the cDNA insert in bothdirections Oligonucleotides synthesis and DNA sequencingwere performed by Eurofins MWG Operon (EbersbergGermany) Each mutant protein was expressed in an E coliHMS174(120582DE3) and purification was done by the sameprocedure published previously [23] and resulted in high

yields of gt95 pure enzymes that exhibited the size of wildtype rcSHMT

43 Spectra and Kinetic Studies All spectra and steady statekinetic studies were performed in a cell with a path lengthof 1 cm with an Agilent 8354 spectrophotometer at 30∘C in a20mM potassium phosphate buffer pH 73 containing 5mM2-mercaptoethanol and 02mM ethylenediaminetetraaceticacid Kinetic assays were performed as previously reported[23] Briefly catalytic assay for L-serine and H

4PteGlu

was measured by coupling the product CH2-H4PteGlu to

methylenetetrahydrofolate dehydrogenase with the concomi-tant reduction of NADP toNADPH To determine the119870

119898for

L-serine H4PteGlu was maintained at 015mM and the L-

serine concentrationwas varied between 005mMand 5mMThe concentration of mutant rcSHMT in these assays was10 120583M The rate of L-allo-threonine cleavage was assayedby coupling the reduction of the product acetaldehyde withNADH and alcohol dehydrogenase Kinetic constants valueswere determined fromdouble-reciprocal plots of the decreasein absorbance at 340 nm with L-allo-threonine concentra-tions varied between 05 and 50mM

44 Rapid Reaction Studies Stopped-flow absorbance exper-iments were performed with an Applied Photophysics SX18apparatus (Leatherhead UK) equipped with a 1 cm opticalpath observation chamber Temperatures were held at either8∘C by a circulating water bath Each study was an average of4ndash6 traces

The rate of formation of the gem-diamine intermediatewas measured by following the increase at 343 nm for thefirst 04 seconds after mixing enzyme and substrate solutionsThe effect of substrate concentration was determined byvarying L-serine and glycine concentrations in a 05ndash5mMrange The curves for absorbance variation versus time werefit by a single-exponential curve For each concentrationof substrate both the first-order rate constant 119896obs andthe amplitude of the spectra change were determined Rateconstants and amplitudes for each individual reaction variedless than 10 from the average values for each reaction

For calculation of 119896on and 119896off the following equation wasapplied assuming the reaction to be pseudo-first toward [119864]and [119878] equal to free substrate concentration

119896obs = 119896on [119878] +119896off (1)

The values of 119896on and 119896off for the rate of formation andbreakdown of the enzyme-substrate complex absorbing at343 nm can be then determined from the slope and 119910-axisintercept of the 119896obs versus [119878] graph A double-reciprocalplot of absorbance changes at 343 nm versus substrate con-centration gives a linear fit with an 119909-axis intercept repre-senting 119870

119889 The enzyme concentration was held constant

at 40 120583M The buffer used was a mix of 20mM potassium2-(N-morpholino) ethanesulfonic acid (MES) 20mM NN-bis[2-hydroxyethyl]-2-aminoethane sulfonate (BES) and20mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid(HEPES) brought to pH 64 68 72 76 and 80 Theconcentrations of the anionic forms of amino acids were


calculated using the Henderson-Hasselbalch equation andpK values of 96 and 92 for the amino groups of glycine andL-serine respectively

45 Crystallization All forms of the rcSHMT (50mgmL)and its mutants and complexes were crystallized aspreviously reported [20] from 2-3 PEG4000 20mMK2HPO4KH2PO4 and 50mM potassium MES or sodium

HEPES (pH 70) in hanging drops or in 05mL Eppendorftubes at room temperature The complexes with glycine andserine had 60mM of the amino acid in the crystallizationdrop

451 Data Collection and Structure Determination Crystalswere transferred to a stabilization solution of 48 polyethy-lene glycol 4000 in the same buffer for 1 hour then transiently(lt30 sec) placed in a cryoprotectant of 30 polyethyleneglycol 400 and 6 polyethylene glycol 4000 in 50mMpotassium MES (pH 70) and flash-frozen in liquid N

2for

data collection Data for a 100∘ sector were collected on aRAxisII with Osmics confocal optics at 60 kV and 150mAOscillation frames were integrated with Denzo and mergedwith Scalepack [29] Merged intensity data were convertedto structure factor amplitudes using Truncate refmac5 [30]All crystals except the T254C-glycine were indexed in spacegroup P4

1with a tetramer per asymmetric unit T254C-

glycine was indexed in space group P41212 with one dimer

per asymmetric unit Structures were solved by molecularreplacement using a rabbit cytosolic SHMT dimer from pdbentry 1CJ0 as a search model and refined using alternatingcycles ofmanual fitting into SigmaAweighted 2mFodFc mapsin COOT [31] and computational refinement in CNS [32]and refmac5 [30] Structure factors and coordinates are beingdeposited to the RCSB protein databank

Abbreviations

SHMT Serine hydroxymethyltransferaserc Rabbit cytosolicmc Mouse cytosolicec Escherichia colibs Bacillus stearothermophilusPLP Pyridoxal 51015840-phosphategem-diamine Geminal diamineH4PteGlu Tetrahydrofolate

(tetrahydropteroylglutamate)510-CH

2-H4PteGlu 510-Methylenetetrahydrofolate

510-CH+=H4PteGlu 510-Methenyltetrahydrofolate

5-CHO-H4PteGlu 5-Formyltetrahydrofolate

Acknowledgments

This work was supported by the National Institutes of Healththrough a Grant (Grant No CA 16059-28) to the MasseyCancer Center of Virginia Commonwealth University insupport of the structural biology resources used in this studyand by grants from the Italian Ministero dellrsquoIstruzione

dellrsquoUniversita e della Ricerca and from FinanziamentoProgetti di Ricerca 2011 of Sapienza University of Rome

References

[1] V Schirch Mechanism of Folate-Requiring Enzymes in One-Carbon Metabolism vol 1 Academic Press San Diego CalifUSA 1998

[2] R Florio M L Di Salvo M Vivoli and R Contestabile ldquoSerinehydroxymethyltransferase a model enzyme for mechanisticstructural and evolutionary studiesrdquo Biochimica et BiophysicaActa vol 1814 no 11 pp 1489ndash1495 2011

[3] A Amadasi M Bertoldi R Contestabile et al ldquoPyridoxal 51015840-phosphate enzymes as targets for therapeutic agentsrdquo CurrentMedicinal Chemistry vol 14 no 12 pp 1291ndash1324 2007

[4] F Daidone R Florio S Rinaldo et al ldquoIn silico and in vitrovalidation of serine hydroxymethyltransferase as a chemother-apeutic target of the antifolate drug pemetrexedrdquo EuropeanJournal of Medicinal Chemistry vol 46 no 5 pp 1616ndash16212011

[5] M L di Salvo R Contestabile A Paiardini and B MarasldquoGlycine consumption and mitochondrial serine hydrox-ymethyltransferase in cancer cells the heme connectionrdquoMed-ical Hypotheses vol 80 pp 633ndash636 2013

[6] P Stover and V Schirch ldquoEnzymatic mechanism for thehydrolysis of 510-methenyltetrahydropteroylglutamate to 5-formyltetrahydropteroylglutamate by serine hydroxymethyl-transferaserdquo Biochemistry vol 31 no 7 pp 2155ndash2164 1992

[7] M L di Salvo R Florio A Paiardini M Vivoli S DrsquoAguannoand R Contestabile ldquoAlanine racemase from Tolypocladiuminflatum a key PLP-dependent enzyme in cyclosporin biosyn-thesis and a model of catalytic promiscuityrdquo Archives of Bio-chemistry and Biophysics vol 529 pp 55ndash65 2013

[8] R Contestabile A Paiardini S Pascarella M L Di Salvo SDrsquoAguanno and F Bossa ldquoL-Threonine aldolase serine hydrox-ymethyltransferase and fungal alanine racemase a subgroupof strictly related enzymes specialized for different functionsrdquoEuropean Journal of Biochemistry vol 268 no 24 pp 6508ndash6525 2001

[9] V Schirch and D M E Szebenyi ldquoSerine hydroxymethyltrans-ferase revisitedrdquoCurrent Opinion in Chemical Biology vol 9 no5 pp 482ndash487 2005

[10] G Giardina R Montioli S Gianni et al ldquoOpen conformationof human DOPA decarboxylase reveals the mechanism ofPLP addition to Group II decarboxylasesrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 108 no 51 pp 20514ndash20519 2011

[11] F Malerba A Bellelli A Giorgi F Bossa and R Contesta-bile ldquoThe mechanism of addition of pyridoxal 51015840-phosphateto Escherichia coli apo-serine hydroxymethyltransferaserdquo Bio-chemical Journal vol 404 no 3 pp 477ndash485 2007

[12] L Schirch ldquoSerine transhydroxymethylase relaxation and tran-sient kinetic study of the formation and interconversion of theenzyme glycine complexesrdquo Journal of Biological Chemistry vol250 no 5 pp 1939ndash1945 1975

[13] C-F Cheng and J L Haslam ldquoA kinetic investigation ofthe interaction of serine transhydroxymethylase with glycinerdquoBiochemistry vol 11 no 19 pp 3512ndash3518 1972

[14] H C Dunathan ldquoConformation and reaction specificity inpyridoxal phosphate enzymesrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 55 no4 pp 712ndash716 1966


[15] L Schirch ldquoSerine hydroxymethyltransferaserdquo Advances inEnzymology and Related Areas of Molecular Biology vol 53 pp83ndash112 1982

[16] S Pascarella S Angelaccio R Contestabile S Delle Fratte MDi Salvo and F Bossa ldquoThe structure of serine hydroxymethyl-transferase as modeled by homology and validated by site-directed mutagenesisrdquo Protein Science vol 7 no 9 pp 1976ndash1982 1998

[17] S Angelaccio S Pascarella E Fattori F BossaW Strong andVSchirch ldquoSerine hydroxymethyltransferase origin of substratespecificityrdquo Biochemistry vol 31 no 1 pp 155ndash162 1992

[18] M Vivoli F Angelucci A Ilari et al ldquoRole of a conservedactive site cation-120587 interaction in Escherichia coli serine hydrox-ymethyltransferaserdquo Biochemistry vol 48 no 50 pp 12034ndash12046 2009

[19] P Baiocco L J Gourlay F Angelucci et al ldquoProbing theMechanism of GSH Activation in Schistosoma haematobiumGlutathione-S-transferase by Site-directed Mutagenesis and X-ray Crystallographyrdquo Journal of Molecular Biology vol 360 no3 pp 678ndash689 2006

[20] J N Scarsdale G Kazanina S Radaev V Schirch and H TWright ldquoCrystal structure of rabbit cytosolic serine hydrox-ymethyltransferase at 28 A resolution mechanistic implica-tionsrdquo Biochemistry vol 38 no 26 pp 8347ndash8358 1999

[21] J M Goldberg J Zheng H Deng Y Q Chen R Callenderand J F Kirsch ldquoStructure of the complex between pyridoxal51015840-phosphate and the tyrosine 225 to phenylalanine mutantof Escherichia coli aspartate aminotransferase determined byisotope-edited classical Raman difference spectroscopyrdquo Bio-chemistry vol 32 no 32 pp 8092ndash8097 1993

[22] V Trivedi A Gupta V R Jala et al ldquoCrystal structure of binaryand ternary complexes of serine hydroxymethyltransferasefrom Bacillus stearothermophilus Insights into the catalyticmechanismrdquo Journal of Biological Chemistry vol 277 no 19 pp17161ndash17169 2002

[23] D M E Szebenyi F N Musayev M L Di Salvo M K SafoandV Schirch ldquoSerine hydroxymethyltransferase role of Glu75and evidence that serine is cleaved by a retroaldol mechanismrdquoBiochemistry vol 43 no 22 pp 6865ndash6876 2004

[24] T-F Fu J N Scarsdale G Kazanina V Schirch and H TWright ldquoLocation of the pteroylpolyglutamate-binding site onrabbit cytosolic serine hydroxymethyltransferaserdquo Journal ofBiological Chemistry vol 278 no 4 pp 2645ndash2653 2003

[25] D M E Szebenyi X Liu I A Kriksunov P J Stover andD J Thiel ldquoStructure of a murine cytoplasmic serine hydrox-ymethyltransferase quinonoid ternary complex evidence forasymmetric obligate dimersrdquo Biochemistry vol 39 no 44 pp13313ndash13323 2000

[26] R Contestabile S Angelaccio F Bossa et al ldquoRole of tyrosine65 in the mechanism of serine hydroxymethyltransferaserdquoBiochemistry vol 39 no 25 pp 7492ndash7500 2000

[27] B S Bhavani V Rajaram S Bisht et al ldquoImportance of tyrosineresidues of Bacillus stearothermophilus serine hydroxymethyl-transferase in cofactor binding and l-allo-Thr cleavage crystalstructure and biochemical studiesrdquo FEBS Journal vol 275 no18 pp 4606ndash4619 2008

[28] M L Di Salvo S D Fratte D De Biase F Bossa and VSchirch ldquoPurification and characterization of recombinant rab-bit cytosolic serine hydroxymethyltransferaserdquo Protein Expres-sion and Purification vol 13 no 2 pp 177ndash183 1998

[29] M Otwinowski ldquoProcessing of X-ray diffraction data collectedin oscillation moderdquo in Methods in Enzymology W Charles

and J Carter Eds Macromolecular Crystallography Part A pp307ndash326 Elsevier 1997

[30] ldquoThe CCP4 suite programs for protein crystallographyrdquo ActaCrystallographica D vol 50 pp 760ndash763 1994

[31] P Emsley and K Cowtan ldquoCoot model-building tools formolecular graphicsrdquo Acta Crystallographica D vol 60 no 12pp 2126ndash2132 2004

[32] A T Brunger P D Adams G M Clore et al ldquoCrystallographyamp NMR system a new software suite for macromolecularstructure determinationrdquo Acta Crystallographica D vol 54 no5 pp 905ndash921 1998

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


External aldimine

HHC

H R

NH

HHC

R

H

HR

Internal aldimine

HHC

H R EnzEnz

C

HHC

HR

H

HEnz

H

Enz

N

Hydrated internal aldimine

HN

HC

Enz

HO

Quinonoid intermediate

O

HN

HC

Enz

Internal aldimine

(carbinolamine Amax

asymp 340nm)

H+

2minusO3PO

NH+

2minusO3PO

Ominus

Ominus

Ominus

CH2

NH+

2minusO3POCH2

Lys257

Lys257 Lys257Lys257

Lys257

Lys257

(ketoenamine form Amax

asymp 430nm)

NH+

N+ N+ N+ N+

N+

2minusO3POCH2

COOminus

COOminus COOminus

COOminus

COOminus

NH NH2

(enolimine form Amax

asymp 340nm)

(Amax

asymp 343nm) (Amax

asymp 343nm)(A

maxasymp 426nm)

(Amax

asymp 498nm)

Gem-diamine I

H2 C

HC

NH+

NH+

2minusO3PO 2minusO3PO

2minusO3PO

Ominus

Ominus

Ominus

Gem-diamine II

H2

CH2

CH2

OHminus

N

Scheme 1 Mechanism of transaldimination reaction of SHMT

substrate amino acids the initial step in the catalytic reactionis the formation of the ldquogeminal diaminerdquo (gem-diamine)between the C41015840 aldehyde of PLP and the amino group ofthe substrate The orientation of the substrate in the activesite with respect to the plane of PLP to which it is covalentlylinked through its amino group in turn determines whichof the three substituent bonds on C120572 of the substrate will becleaved In 1966 Dunathan [14] provided a unifying conceptfor the specific selection of the substrate scissile bond in PLP-dependent enzymes which has been confirmed in solutionand structural studies In his proposal all bonds broken andmade on the catalytic pathway are the nearest perpendicularto the conjugated 120587 system of the PLP ringThe resulting car-banion at C120572 of the substrate amino acid is stabilized by reso-nance with the 120587-electron system in the pyridine ring of PLPThis intermediate on the catalytic pathway is referred to as theldquoquinonoidrdquo complex and absorbs near 500 nm (Scheme 1)Solution studies have shown that SHMT passes throughseveral ordered spectrophotometrically identifiable interme-diates that reflect changes in the electron system of the PLPcofactor [12 15] and are consistent with Dunathanrsquos proposal

Prior to the availability of crystallographic structureinformation on SHMT it was noted that the amino acidsequences of SHMTs from diverse species have a conservedrun of 4 threonine residues terminating 2 residues upstreamof the active site lysine V-V-T-T-T254-T-H-K257(PLP)-T(numbering is that for rabbit cytosolic serine hydroxymethyl-transferase (rcSHMT)) [16] To determine the possible rolesof this conserved sequence in SHMTcatalysis each threonineof this active site stretch was mutated in ecSHMT to analanine and the effects of the changes on the spectral andkinetic properties were investigated [17] It was found that

only the T226A mutant of ecSHMT (Thr226 in ecSHMTnumbering is equivalent to rcSHMTThr254) had significantspectral and kinetic differences from the wild type enzymeThere was a 32-fold lower 119896cat in the conversion of L-serine toglycine and the T226A mutant was virtually inactive towardcleavage of L-allo-threonine compared to wild type ecSHMTFurthermore in the presence of L-serine the T226A mutantexhibited a large spectral absorbance peak at 343 nm whichis characteristic of a gem-diamine intermediate and only asmall peak at 425 nm characteristic of the external aldimineStopped-flow analysis showed that the 343 nm peak wasformed rapidly but its conversion to the 425 nm absorbingpeak was slow [17] Since the gem-diamine is generally ashort-lived intermediate on the reaction pathways of PLP-dependent enzymes this T226A ecSHMTmutant offered theopportunity to investigate the structural changes that appar-ently slowed the conversion of the gem-diamine intermediateto the external aldimine We were unable to obtain crystalsfor the T226A mutant of ecSHMT However we were ableto crystallize the homologous T254A and T254C mutants ofrcSHMT We report here the kinetic properties and crystalstructures of the T254 mutants of rcSHMT and their glycineand L-serine complexes In addition we have increased theresolution of the structure of wild type rcSHMT to 21 A as anaid in examining details of the gem-diamine structures

2 Results and Discussion

21 Spectroscopic Studies Wild type rcSHMT exhibits acharacteristic major single absorption band with maximumintensity at 430 nm due to the protonated internal aldiminebetween enzyme and cofactor Also aminor band is observed




4PteGlu to




















000

004

008

012

016

Wavelength (nm)300 350 400 450 500

Wild type

+gly

+gly

No ligand

Abso

rban

ce

+H4PteGlu

(a)

Wavelength (nm)300 350 400 450 500 550

Wild type

+L-ser

No ligand

000

004

008

012

016

Abso

rban

ce

(b)

000

002

004

006

008

Wavelength (nm)300 350 400 450 500

T254A

+gly

+gly

No ligand

Abso

rban

ce

+H4PteGlu

(c)

Wavelength (nm)300 350 400 450 500 550

T254A

+L-ser

No ligand

000

002

004

006

008

Abso

rban

ce

(d)

Wavelength (nm)300 350 400 450 500

000

002

004

006

008T254C

+gly

No ligand

Abso

rban

ce

+gly+H4PteGlu

(e)

Wavelength (nm)300 350 400 450 500 550

T254C

+L-ser

No ligand

000

002

004

006

008

Abso

rban

ce

(f)








0

5

10

15

20

25

60 65 70 75 80 85 90pH

T254A

k998400 on

(sminus1

Mminus1times106)

(a)

60 65 70 75 80 85 900

5

10

15

20

pH

T254C

k998400 on

(sminus1

Mminus1times106)

(b)





















1015840


minus1)

L-Serine

64 33 20 103 29 18 39

68 58 14 83 40 10 36

72 87 86 58 68 67 32

76 95 37 53 87 34 27

80 107 18 31 94 15 23

Glycine

64 08 13 10 05 85 51

68 13 80 13 07 45 67

72 21 50 19 15 38 69

76 30 29 24 21 22 70

80 44 18 29 37 16 68









Table3Cr

ystallo

graphicd

atac

ollectionandrefin

ementstatistic

sfor

rcSH

MTstr

ucturesd

etermined

inthiswork

Wild

type

T254A

T254A+gly

T254A+L-ser

T254C

T254C+gly

T254C+L-ser

Cellspace

grou

p115611561560

P41

115111511574

P41

115011501568

P41

115511551563

P41

114011401547

P41

114611461569

P412

12114

011401556

P41

Resolutio

n119

0ndash210

(216

ndash210

)1118



115ndash240

(246ndash

240

)1118

ndash240(246ndash

240


1118


Com

pleteness()

985(982)

922(879

)996(998

)883(858)

901(869)

696(620)

909(769)

119877work

199(2

48)

201

(306)

203

(292)

197(2

47)

195(2

42)

221

(333)

202

(284)

119877fre

e242

(316)

271

(432)

277

(399)

258

(318)

256

(346

)305

(414)

263

(362)

119873work

105631

(766

8)55295(3849)

52905(3769)

63363(4478)

62364(4393)

19976(1244)

52787(5963)

119873fre

e117

36(845)

6204

(426)

5968

(406)

7156

(510)

7009

(470)

1654

(96)

5963

(375)

⟨119861protein⟩

308

448

34373

394

366

38⟨119861water⟩

398

438

346

373

419

NA

401

RMSD

from

ideal

Bond

leng

ths

0008

0007

0007

000

6000

6000

60007

Bond

angles

1312

412

2113

113

108

116

Ramachand

ranplot

Favored

1460

1424

1403

1425

1435

702

1427

Additio

nally

allowed

123

142

142

146

133

69128

Generou

slyallowed

138

912

156

12Fo

rbidden

37

95

57

10MolProb

ityscore

173(93

)204

(96

)204

(97

)18

8(96

)18

1(97)

224

(93

)200

(96

)


Thr254Ala254

Lys257

(a)

Thr254Ala254

Lys257

(b)

Thr254Ala254

Lys257

(c)

Thr254Ala254

Lys257

(d)











Ala254

Glu75

Tyr83

Arg402

Ser203

His231

Lys257

(a)

Ala254

Glu75

Tyr83

Arg402

Ser203

His231

Lys257

(b)


Tyr73 Tyr83

Arg402

Thr254Ala254









Tyr73 Tyr83

Arg402

Lys257

(a)

Tyr73 Tyr83

Arg402

Lys257

(b)



3 Conclusions






2





4PteGlu







A ia PO42minus

B ia PO42minus

C ia MESD ia MES

Unliganded T254A

A ia PO42minus

B ia PO42minus

C ia PO42minus

D ia PO42minus

T254A + glycine


T254A + L-serine


Unliganded T254C



T254C + L-serine








4PteGlu



119898for






119896obs = 119896on [119878] +119896off (1)









2for





Abbreviations






Acknowledgments



References










































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




4PteGlu to




















000

004

008

012

016

Wavelength (nm)300 350 400 450 500

Wild type

+gly

+gly

No ligand

Abso

rban

ce

+H4PteGlu

(a)

Wavelength (nm)300 350 400 450 500 550

Wild type

+L-ser

No ligand

000

004

008

012

016

Abso

rban

ce

(b)

000

002

004

006

008

Wavelength (nm)300 350 400 450 500

T254A

+gly

+gly

No ligand

Abso

rban

ce

+H4PteGlu

(c)

Wavelength (nm)300 350 400 450 500 550

T254A

+L-ser

No ligand

000

002

004

006

008

Abso

rban

ce

(d)

Wavelength (nm)300 350 400 450 500

000

002

004

006

008T254C

+gly

No ligand

Abso

rban

ce

+gly+H4PteGlu

(e)

Wavelength (nm)300 350 400 450 500 550

T254C

+L-ser

No ligand

000

002

004

006

008

Abso

rban

ce

(f)








0

5

10

15

20

25

60 65 70 75 80 85 90pH

T254A

k998400 on

(sminus1

Mminus1times106)

(a)

60 65 70 75 80 85 900

5

10

15

20

pH

T254C

k998400 on

(sminus1

Mminus1times106)

(b)





















1015840


minus1)

L-Serine

64 33 20 103 29 18 39

68 58 14 83 40 10 36

72 87 86 58 68 67 32

76 95 37 53 87 34 27

80 107 18 31 94 15 23

Glycine

64 08 13 10 05 85 51

68 13 80 13 07 45 67

72 21 50 19 15 38 69

76 30 29 24 21 22 70

80 44 18 29 37 16 68









Table3Cr

ystallo

graphicd

atac

ollectionandrefin

ementstatistic

sfor

rcSH

MTstr

ucturesd

etermined

inthiswork

Wild

type

T254A

T254A+gly

T254A+L-ser

T254C

T254C+gly

T254C+L-ser

Cellspace

grou

p115611561560

P41

115111511574

P41

115011501568

P41

115511551563

P41

114011401547

P41

114611461569

P412

12114

011401556

P41

Resolutio

n119

0ndash210

(216

ndash210

)1118



115ndash240

(246ndash

240

)1118

ndash240(246ndash

240


1118


Com

pleteness()

985(982)

922(879

)996(998

)883(858)

901(869)

696(620)

909(769)

119877work

199(2

48)

201

(306)

203

(292)

197(2

47)

195(2

42)

221

(333)

202

(284)

119877fre

e242

(316)

271

(432)

277

(399)

258

(318)

256

(346

)305

(414)

263

(362)

119873work

105631

(766

8)55295(3849)

52905(3769)

63363(4478)

62364(4393)

19976(1244)

52787(5963)

119873fre

e117

36(845)

6204

(426)

5968

(406)

7156

(510)

7009

(470)

1654

(96)

5963

(375)

⟨119861protein⟩

308

448

34373

394

366

38⟨119861water⟩

398

438

346

373

419

NA

401

RMSD

from

ideal

Bond

leng

ths

0008

0007

0007

000

6000

6000

60007

Bond

angles

1312

412

2113

113

108

116

Ramachand

ranplot

Favored

1460

1424

1403

1425

1435

702

1427

Additio

nally

allowed

123

142

142

146

133

69128

Generou

slyallowed

138

912

156

12Fo

rbidden

37

95

57

10MolProb

ityscore

173(93

)204

(96

)204

(97

)18

8(96

)18

1(97)

224

(93

)200

(96

)


Thr254Ala254

Lys257

(a)

Thr254Ala254

Lys257

(b)

Thr254Ala254

Lys257

(c)

Thr254Ala254

Lys257

(d)











Ala254

Glu75

Tyr83

Arg402

Ser203

His231

Lys257

(a)

Ala254

Glu75

Tyr83

Arg402

Ser203

His231

Lys257

(b)


Tyr73 Tyr83

Arg402

Thr254Ala254









Tyr73 Tyr83

Arg402

Lys257

(a)

Tyr73 Tyr83

Arg402

Lys257

(b)



3 Conclusions






2





4PteGlu







A ia PO42minus

B ia PO42minus

C ia MESD ia MES

Unliganded T254A

A ia PO42minus

B ia PO42minus

C ia PO42minus

D ia PO42minus

T254A + glycine


T254A + L-serine


Unliganded T254C



T254C + L-serine








4PteGlu



119898for






119896obs = 119896on [119878] +119896off (1)









2for





Abbreviations






Acknowledgments



References










































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


000

004

008

012

016

Wavelength (nm)300 350 400 450 500

Wild type

+gly

+gly

No ligand

Abso

rban

ce

+H4PteGlu

(a)

Wavelength (nm)300 350 400 450 500 550

Wild type

+L-ser

No ligand

000

004

008

012

016

Abso

rban

ce

(b)

000

002

004

006

008

Wavelength (nm)300 350 400 450 500

T254A

+gly

+gly

No ligand

Abso

rban

ce

+H4PteGlu

(c)

Wavelength (nm)300 350 400 450 500 550

T254A

+L-ser

No ligand

000

002

004

006

008

Abso

rban

ce

(d)

Wavelength (nm)300 350 400 450 500

000

002

004

006

008T254C

+gly

No ligand

Abso

rban

ce

+gly+H4PteGlu

(e)

Wavelength (nm)300 350 400 450 500 550

T254C

+L-ser

No ligand

000

002

004

006

008

Abso

rban

ce

(f)








0

5

10

15

20

25

60 65 70 75 80 85 90pH

T254A

k998400 on

(sminus1

Mminus1times106)

(a)

60 65 70 75 80 85 900

5

10

15

20

pH

T254C

k998400 on

(sminus1

Mminus1times106)

(b)





















1015840


minus1)

L-Serine

64 33 20 103 29 18 39

68 58 14 83 40 10 36

72 87 86 58 68 67 32

76 95 37 53 87 34 27

80 107 18 31 94 15 23

Glycine

64 08 13 10 05 85 51

68 13 80 13 07 45 67

72 21 50 19 15 38 69

76 30 29 24 21 22 70

80 44 18 29 37 16 68









Table3Cr

ystallo

graphicd

atac

ollectionandrefin

ementstatistic

sfor

rcSH

MTstr

ucturesd

etermined

inthiswork

Wild

type

T254A

T254A+gly

T254A+L-ser

T254C

T254C+gly

T254C+L-ser

Cellspace

grou

p115611561560

P41

115111511574

P41

115011501568

P41

115511551563

P41

114011401547

P41

114611461569

P412

12114

011401556

P41

Resolutio

n119

0ndash210

(216

ndash210

)1118



115ndash240

(246ndash

240

)1118

ndash240(246ndash

240


1118


Com

pleteness()

985(982)

922(879

)996(998

)883(858)

901(869)

696(620)

909(769)

119877work

199(2

48)

201

(306)

203

(292)

197(2

47)

195(2

42)

221

(333)

202

(284)

119877fre

e242

(316)

271

(432)

277

(399)

258

(318)

256

(346

)305

(414)

263

(362)

119873work

105631

(766

8)55295(3849)

52905(3769)

63363(4478)

62364(4393)

19976(1244)

52787(5963)

119873fre

e117

36(845)

6204

(426)

5968

(406)

7156

(510)

7009

(470)

1654

(96)

5963

(375)

⟨119861protein⟩

308

448

34373

394

366

38⟨119861water⟩

398

438

346

373

419

NA

401

RMSD

from

ideal

Bond

leng

ths

0008

0007

0007

000

6000

6000

60007

Bond

angles

1312

412

2113

113

108

116

Ramachand

ranplot

Favored

1460

1424

1403

1425

1435

702

1427

Additio

nally

allowed

123

142

142

146

133

69128

Generou

slyallowed

138

912

156

12Fo

rbidden

37

95

57

10MolProb

ityscore

173(93

)204

(96

)204

(97

)18

8(96

)18

1(97)

224

(93

)200

(96

)


Thr254Ala254

Lys257

(a)

Thr254Ala254

Lys257

(b)

Thr254Ala254

Lys257

(c)

Thr254Ala254

Lys257

(d)











Ala254

Glu75

Tyr83

Arg402

Ser203

His231

Lys257

(a)

Ala254

Glu75

Tyr83

Arg402

Ser203

His231

Lys257

(b)


Tyr73 Tyr83

Arg402

Thr254Ala254









Tyr73 Tyr83

Arg402

Lys257

(a)

Tyr73 Tyr83

Arg402

Lys257

(b)



3 Conclusions






2





4PteGlu







A ia PO42minus

B ia PO42minus

C ia MESD ia MES

Unliganded T254A

A ia PO42minus

B ia PO42minus

C ia PO42minus

D ia PO42minus

T254A + glycine


T254A + L-serine


Unliganded T254C



T254C + L-serine








4PteGlu



119898for






119896obs = 119896on [119878] +119896off (1)









2for





Abbreviations






Acknowledgments



References










































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


0

5

10

15

20

25

60 65 70 75 80 85 90pH

T254A

k998400 on

(sminus1

Mminus1times106)

(a)

60 65 70 75 80 85 900

5

10

15

20

pH

T254C

k998400 on

(sminus1

Mminus1times106)

(b)





















1015840


minus1)

L-Serine

64 33 20 103 29 18 39

68 58 14 83 40 10 36

72 87 86 58 68 67 32

76 95 37 53 87 34 27

80 107 18 31 94 15 23

Glycine

64 08 13 10 05 85 51

68 13 80 13 07 45 67

72 21 50 19 15 38 69

76 30 29 24 21 22 70

80 44 18 29 37 16 68









Table3Cr

ystallo

graphicd

atac

ollectionandrefin

ementstatistic

sfor

rcSH

MTstr

ucturesd

etermined

inthiswork

Wild

type

T254A

T254A+gly

T254A+L-ser

T254C

T254C+gly

T254C+L-ser

Cellspace

grou

p115611561560

P41

115111511574

P41

115011501568

P41

115511551563

P41

114011401547

P41

114611461569

P412

12114

011401556

P41

Resolutio

n119

0ndash210

(216

ndash210

)1118



115ndash240

(246ndash

240

)1118

ndash240(246ndash

240


1118


Com

pleteness()

985(982)

922(879

)996(998

)883(858)

901(869)

696(620)

909(769)

119877work

199(2

48)

201

(306)

203

(292)

197(2

47)

195(2

42)

221

(333)

202

(284)

119877fre

e242

(316)

271

(432)

277

(399)

258

(318)

256

(346

)305

(414)

263

(362)

119873work

105631

(766

8)55295(3849)

52905(3769)

63363(4478)

62364(4393)

19976(1244)

52787(5963)

119873fre

e117

36(845)

6204

(426)

5968

(406)

7156

(510)

7009

(470)

1654

(96)

5963

(375)

⟨119861protein⟩

308

448

34373

394

366

38⟨119861water⟩

398

438

346

373

419

NA

401

RMSD

from

ideal

Bond

leng

ths

0008

0007

0007

000

6000

6000

60007

Bond

angles

1312

412

2113

113

108

116

Ramachand

ranplot

Favored

1460

1424

1403

1425

1435

702

1427

Additio

nally

allowed

123

142

142

146

133

69128

Generou

slyallowed

138

912

156

12Fo

rbidden

37

95

57

10MolProb

ityscore

173(93

)204

(96

)204

(97

)18

8(96

)18

1(97)

224

(93

)200

(96

)


Thr254Ala254

Lys257

(a)

Thr254Ala254

Lys257

(b)

Thr254Ala254

Lys257

(c)

Thr254Ala254

Lys257

(d)











Ala254

Glu75

Tyr83

Arg402

Ser203

His231

Lys257

(a)

Ala254

Glu75

Tyr83

Arg402

Ser203

His231

Lys257

(b)


Tyr73 Tyr83

Arg402

Thr254Ala254









Tyr73 Tyr83

Arg402

Lys257

(a)

Tyr73 Tyr83

Arg402

Lys257

(b)



3 Conclusions






2





4PteGlu







A ia PO42minus

B ia PO42minus

C ia MESD ia MES

Unliganded T254A

A ia PO42minus

B ia PO42minus

C ia PO42minus

D ia PO42minus

T254A + glycine


T254A + L-serine


Unliganded T254C



T254C + L-serine








4PteGlu



119898for






119896obs = 119896on [119878] +119896off (1)









2for





Abbreviations






Acknowledgments



References










































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology







1015840


minus1)

L-Serine

64 33 20 103 29 18 39

68 58 14 83 40 10 36

72 87 86 58 68 67 32

76 95 37 53 87 34 27

80 107 18 31 94 15 23

Glycine

64 08 13 10 05 85 51

68 13 80 13 07 45 67

72 21 50 19 15 38 69

76 30 29 24 21 22 70

80 44 18 29 37 16 68









Table3Cr

ystallo

graphicd

atac

ollectionandrefin

ementstatistic

sfor

rcSH

MTstr

ucturesd

etermined

inthiswork

Wild

type

T254A

T254A+gly

T254A+L-ser

T254C

T254C+gly

T254C+L-ser

Cellspace

grou

p115611561560

P41

115111511574

P41

115011501568

P41

115511551563

P41

114011401547

P41

114611461569

P412

12114

011401556

P41

Resolutio

n119

0ndash210

(216

ndash210

)1118



115ndash240

(246ndash

240

)1118

ndash240(246ndash

240


1118


Com

pleteness()

985(982)

922(879

)996(998

)883(858)

901(869)

696(620)

909(769)

119877work

199(2

48)

201

(306)

203

(292)

197(2

47)

195(2

42)

221

(333)

202

(284)

119877fre

e242

(316)

271

(432)

277

(399)

258

(318)

256

(346

)305

(414)

263

(362)

119873work

105631

(766

8)55295(3849)

52905(3769)

63363(4478)

62364(4393)

19976(1244)

52787(5963)

119873fre

e117

36(845)

6204

(426)

5968

(406)

7156

(510)

7009

(470)

1654

(96)

5963

(375)

⟨119861protein⟩

308

448

34373

394

366

38⟨119861water⟩

398

438

346

373

419

NA

401

RMSD

from

ideal

Bond

leng

ths

0008

0007

0007

000

6000

6000

60007

Bond

angles

1312

412

2113

113

108

116

Ramachand

ranplot

Favored

1460

1424

1403

1425

1435

702

1427

Additio

nally

allowed

123

142

142

146

133

69128

Generou

slyallowed

138

912

156

12Fo

rbidden

37

95

57

10MolProb

ityscore

173(93

)204

(96

)204

(97

)18

8(96

)18

1(97)

224

(93

)200

(96

)


Thr254Ala254

Lys257

(a)

Thr254Ala254

Lys257

(b)

Thr254Ala254

Lys257

(c)

Thr254Ala254

Lys257

(d)











Ala254

Glu75

Tyr83

Arg402

Ser203

His231

Lys257

(a)

Ala254

Glu75

Tyr83

Arg402

Ser203

His231

Lys257

(b)


Tyr73 Tyr83

Arg402

Thr254Ala254









Tyr73 Tyr83

Arg402

Lys257

(a)

Tyr73 Tyr83

Arg402

Lys257

(b)



3 Conclusions






2





4PteGlu







A ia PO42minus

B ia PO42minus

C ia MESD ia MES

Unliganded T254A

A ia PO42minus

B ia PO42minus

C ia PO42minus

D ia PO42minus

T254A + glycine


T254A + L-serine


Unliganded T254C



T254C + L-serine








4PteGlu



119898for






119896obs = 119896on [119878] +119896off (1)









2for





Abbreviations






Acknowledgments



References










































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Table3Cr

ystallo

graphicd

atac

ollectionandrefin

ementstatistic

sfor

rcSH

MTstr

ucturesd

etermined

inthiswork

Wild

type

T254A

T254A+gly

T254A+L-ser

T254C

T254C+gly

T254C+L-ser

Cellspace

grou

p115611561560

P41

115111511574

P41

115011501568

P41

115511551563

P41

114011401547

P41

114611461569

P412

12114

011401556

P41

Resolutio

n119

0ndash210

(216

ndash210

)1118



115ndash240

(246ndash

240

)1118

ndash240(246ndash

240


1118


Com

pleteness()

985(982)

922(879

)996(998

)883(858)

901(869)

696(620)

909(769)

119877work

199(2

48)

201

(306)

203

(292)

197(2

47)

195(2

42)

221

(333)

202

(284)

119877fre

e242

(316)

271

(432)

277

(399)

258

(318)

256

(346

)305

(414)

263

(362)

119873work

105631

(766

8)55295(3849)

52905(3769)

63363(4478)

62364(4393)

19976(1244)

52787(5963)

119873fre

e117

36(845)

6204

(426)

5968

(406)

7156

(510)

7009

(470)

1654

(96)

5963

(375)

⟨119861protein⟩

308

448

34373

394

366

38⟨119861water⟩

398

438

346

373

419

NA

401

RMSD

from

ideal

Bond

leng

ths

0008

0007

0007

000

6000

6000

60007

Bond

angles

1312

412

2113

113

108

116

Ramachand

ranplot

Favored

1460

1424

1403

1425

1435

702

1427

Additio

nally

allowed

123

142

142

146

133

69128

Generou

slyallowed

138

912

156

12Fo

rbidden

37

95

57

10MolProb

ityscore

173(93

)204

(96

)204

(97

)18

8(96

)18

1(97)

224

(93

)200

(96

)


Thr254Ala254

Lys257

(a)

Thr254Ala254

Lys257

(b)

Thr254Ala254

Lys257

(c)

Thr254Ala254

Lys257

(d)











Ala254

Glu75

Tyr83

Arg402

Ser203

His231

Lys257

(a)

Ala254

Glu75

Tyr83

Arg402

Ser203

His231

Lys257

(b)


Tyr73 Tyr83

Arg402

Thr254Ala254









Tyr73 Tyr83

Arg402

Lys257

(a)

Tyr73 Tyr83

Arg402

Lys257

(b)



3 Conclusions






2





4PteGlu







A ia PO42minus

B ia PO42minus

C ia MESD ia MES

Unliganded T254A

A ia PO42minus

B ia PO42minus

C ia PO42minus

D ia PO42minus

T254A + glycine


T254A + L-serine


Unliganded T254C



T254C + L-serine








4PteGlu



119898for






119896obs = 119896on [119878] +119896off (1)









2for





Abbreviations






Acknowledgments



References










































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Thr254Ala254

Lys257

(a)

Thr254Ala254

Lys257

(b)

Thr254Ala254

Lys257

(c)

Thr254Ala254

Lys257

(d)











Ala254

Glu75

Tyr83

Arg402

Ser203

His231

Lys257

(a)

Ala254

Glu75

Tyr83

Arg402

Ser203

His231

Lys257

(b)


Tyr73 Tyr83

Arg402

Thr254Ala254









Tyr73 Tyr83

Arg402

Lys257

(a)

Tyr73 Tyr83

Arg402

Lys257

(b)



3 Conclusions






2





4PteGlu







A ia PO42minus

B ia PO42minus

C ia MESD ia MES

Unliganded T254A

A ia PO42minus

B ia PO42minus

C ia PO42minus

D ia PO42minus

T254A + glycine


T254A + L-serine


Unliganded T254C



T254C + L-serine








4PteGlu



119898for






119896obs = 119896on [119878] +119896off (1)









2for





Abbreviations






Acknowledgments



References










































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Ala254

Glu75

Tyr83

Arg402

Ser203

His231

Lys257

(a)

Ala254

Glu75

Tyr83

Arg402

Ser203

His231

Lys257

(b)


Tyr73 Tyr83

Arg402

Thr254Ala254









Tyr73 Tyr83

Arg402

Lys257

(a)

Tyr73 Tyr83

Arg402

Lys257

(b)



3 Conclusions






2





4PteGlu







A ia PO42minus

B ia PO42minus

C ia MESD ia MES

Unliganded T254A

A ia PO42minus

B ia PO42minus

C ia PO42minus

D ia PO42minus

T254A + glycine


T254A + L-serine


Unliganded T254C



T254C + L-serine








4PteGlu



119898for






119896obs = 119896on [119878] +119896off (1)









2for





Abbreviations






Acknowledgments



References










































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Tyr73 Tyr83

Arg402

Lys257

(a)

Tyr73 Tyr83

Arg402

Lys257

(b)



3 Conclusions






2





4PteGlu







A ia PO42minus

B ia PO42minus

C ia MESD ia MES

Unliganded T254A

A ia PO42minus

B ia PO42minus

C ia PO42minus

D ia PO42minus

T254A + glycine


T254A + L-serine


Unliganded T254C



T254C + L-serine








4PteGlu



119898for






119896obs = 119896on [119878] +119896off (1)









2for





Abbreviations






Acknowledgments



References










































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





A ia PO42minus

B ia PO42minus

C ia MESD ia MES

Unliganded T254A

A ia PO42minus

B ia PO42minus

C ia PO42minus

D ia PO42minus

T254A + glycine


T254A + L-serine


Unliganded T254C



T254C + L-serine








4PteGlu



119898for






119896obs = 119896on [119878] +119896off (1)









2for





Abbreviations






Acknowledgments



References










































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology






2for





Abbreviations






Acknowledgments



References










































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Documents

Research Article Structure-Based Mechanism for Early PLP ...downloads.hindawi.com/journals/bmri/2013/458571.pdf · Research Article Structure-Based Mechanism for Early PLP-Mediated