Biosynthesis of Tetrapyrrole Pigment Precursors1fraction precipitating at (NH4)2SO4 concentrations between 35 and 60% ofsaturation was harvested by centrifugation, redis-solvedin assaybuffer(50

$Page 1: Biosynthesis of Tetrapyrrole Pigment Precursors1fraction precipitating at (NH4)2SO4 concentrations between 35 and 60% ofsaturation was harvested by centrifugation, redis-solvedin assaybuffer(50$
Plant Physiol. (1988) 88, 879-8860032-0889/88/88/0879/08/$0 1.00/0

Biosynthesis of Tetrapyrrole Pigment Precursors1FORMATION AND UTILIZATION OF GLUTAMYL-tRNA FOR b-AMINOLEVULINIC ACIDSYNTHESIS BY ISOLATED ENZYME FRACTIONS FROM CHLORELLA VULGARIS

Received for publication April 11, 1988 and in revised form July 5, 1988

YAEL J. AVISSAR AND SAMUEL I. BEALE*Division ofBiology and Medicine, Brown University, Providence, Rhode Island 02912

ABSTRACT

The universal tetrapyrrole precursor 6-aminolevulinic acid (ALA) isformed from glutamate (Glu) in algae and higher plants. In the postulatedreaction sequence, Glu-tRNA is produced by a Glu-tRNA synthetase,and the product serves as a substrate for a reduction step catalyzed by apyridine nucleotide-requiring Glu-tRNA dehydrogenase. The reducedintermediate is then converted into ALA by a transaminase. An RNAand three enzyme fractions required for ALA formation from Glu havebeen isolated from soluble Chlorella extracts. The recombined fractionscatalyzed ALA production from Glu or Glu-tRNA. The fraction contain-ing the synthetase produced Glu-tRNA from Glu and tRNA in thepresence of ATP and Mg2". The isolated product of this reaction servedas substrate for ALA production by the partially reconstituted enzymesystem lacking the synthetase fraction and incapable of producing ALAfrom Glu. The production of ALA from Glu-tRNA by this partiallyreconstituted system did not require free Glu or ATP, and was not affectedby added ATP. These results show that (a) free Glu-tRNA is an inter-mediate in the formation of ALA from Glu, (b) ATP is required only inthe first step of the reaction sequence, and NADPH only in a later step,(c) Glu-tRNA production is the essential reaction catalyzed by one of theenzyme fractions, (d) this enzyme fraction is active in the absence of theother enzymes and is not required for activity of the others. The specificGlu-tRNA synthetase required for ALA formation has an approximatemolecular weight of 73,000 ± 5,000 as determined by Sephadex G-100gel filtration and native polyacrylamide gel electrophoresis. Other Glu-tRNA synthetases were present in the cell extracts but were ineffectivein the the ALA-forming process.

ALA2 is the universal biosynthetic precursor of the tetrapyr-roles, which include hemes, Chls, and bilins (7). ALA is producedin plants, algae, and some bacteria from the intact carbon skel-eton of Glu (2). Enzyme preparations capable of converting Gluto ALA have been obtained from several higher plants includingbarley (13), maize (14), and wheat (11), and the algae Chlorella(35), Chlamydomonas (34), Cyanidium (36), and Euglena (21).Other cell types yielding cell-free preparations capable ofcatalyz-ing ALA formation from Glu include cyanobacteria and pro-chlorophytes (26), Methanobacterium (12), Clostridium (22) andChlorobium (27).The biochemistry ofALA formation from Glu has been stud-

ied in plants and algae, and the proposed pathway is presented

'Supported by National Science Foundation Grant DMB85-18580.2Abbreviations: ALA, 6-aminolevulinic acid; ALA-pyrrole, 1-methyl-

2-carboxyethyl-3-propionic acid pyrrole; Glu, glutamate; PMSF, phenyl-methylsulfonyl fluoride.

in Figure 1. Cell free extracts capable of converting Glu to ALAhave been fractionated to yield a required RNA fraction and two(15, 33) or three required enzyme fractions (6, 37).

Evidence for the role of Glu-tRNA as an intermediate in thereaction sequence was obtained for cell free extracts from Chla-mydomonas (15) and from greening barley (16), using cell extractfractions containing a mixture of two of the proposed enzymeactivities participating in the reaction sequence, Glu-tRNA syn-thetase and Glu-tRNA dehydrogenase. The RNA fraction re-quired for ALA formation has been isolated from several sourcesand characterized as a tRNA bearing the UUC (Glu) anticodon(28, 29). The three enzyme fractions isolated from Chlorellaextracts have been tentatively identified as containing the syn-thetase, dehydrogenase, and the transaminase of the proposedpathway, based primarily on their separation by affinity resinsbearing ligands directed against the proposed cofactor require-ments of each enzyme (37).We now have obtained direct evidence that the aminoacyl-

tRNA synthetase reaction is an essential step and have verifiedthe proposed role ofGlu-tRNA as an intermediate in the reactionsequence leading from Glu to ALA by completely separating thereactions leading to the formation ofGlu-tRNA from those thatsubsequently transform Glu-tRNA to ALA. The two reactionsequences are catalyzed by distinct enzyme and cofactor sets thuspermitting the assignment of specific roles to each. A preliminaryaccount of this work has appeared in abstract form (1).

MATERIALS AND METHODS

Growth of Algae. Chlorella vulgaris Beijerinck strain C- I0 wasgrown in batch culture in a glucose-based liquid medium aspreviously described (35), on rotary shakers at 25°C. Strain C-lOforms Chl only in the light. Cells were grown in completedarkness until late exponential growth phase and then illumi-nated (32 ,uE m-2 s-', supplied by equal numbers of cool-whiteand red fluorescent tubes) for 3 h before harvesting to inducerapid greening.Enzyme Extraction and Purification. Cells were harvested as

previously described (37), resuspended in homogenization buffer(0.1 M Tricine [pH 7.9], 0.3 M glycerol, 15 mM MgCl2, 1 mMDTT, 2 mm EDTA, and 2.5 ppm PMSF), and broken by passagethrough a French pressure cell at 23,000 p.s.i. The homogenatewas centrifuged at 10,000g for 10 min to remove cell debris andunbroken cells, stirred with 0.5 M NaCl on ice for 20 min, andthen centrifuged at 264,000g at 2°C for 90 min. The clear upperportion of the supernatant was strained through glass wool andfractionated by differential (NH4)2SO4 precipitation at 0°C. Thefraction precipitating at (NH4)2SO4 concentrations between 35and 60% of saturation was harvested by centrifugation, redis-solved in assay buffer (50 mm Tricine [pH 7.9], 1 M glycerol, 15mM MgCl2, and 1 mm DTT), and desalted by passage throughSephadex G-25 that had been preequilibrated with assay buffer.

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COOH COOH COOH COOHCH2 0H2 0H2 CH2CH2 ATP, tRNA CH2 NADPH 0H2 PALP 0H2HNH2 Glu-tRNA Synthetase CHNH2 Dehydrogenase HNH2 Transaminase =0

OOOH 00-tRNA CHO CH2NH2

Glu Glu-tRNA [GSA] ALA

FIG. 1. Proposed biosynthetic sequence of ALA formation from Glu via the RNA-dependent five-carbon pathway. The cofactor requirements,enzyme activities, and intermediate products are illustrated. Glutamate-1 -semialdehyde (GSA), the reduced intermediate illustrated, is one of severalproposed ALA precursors.

Fractionation of this enzyme extract on Reactive Blue 2-Sepha-rose was carried out as previously described (37). The unboundfraction (Blu-1) was precipitated by addition of (NH4)2SO4 to70% of saturating concentration, the precipitate was redissolvedin assay buffer, desalted by passage through Sephadex G-25, andstored in aliquots at -75°C. The fraction eluted with 0.1 M NaCl(Blu-2) was discarded. The fraction eluted with 0.5 M NaCl (Blu-3) was concentrated and desalted as described for fraction Blu-1,then supplemented with EDTA to 5 mM and applied to a columnof 2',5'-ADP-agarose as described previously (37). The unboundfraction (A-1) was precipitated by addition of (NH4)2SO4 to 90%of saturating concentration, the precipitate was redissolved inassay buffer, desalted by passage through Sephadex G-25, andstored in aliquots at -75°C. The column was then eluted with25 mL of 1 mM NADH, yielding fraction A-2, and finally with25 mL of 1 mm NADPH, yielding fraction A-3. Fractions A-2and A-3 were precipitated with (NH4)2SO4, redissolved, desalted,and stored as described for fraction A- 1.RNA Extraction and Partial Purification. RNA was prepared

as previously described (37). To obtain RNA, the lower, pig-mented portion of the high-speed supernatant remaining afterthe removal of the clear upper portion for enzyme fractionationwas combined with the supernatant fraction remaining afterprecipitation of the enzymes at 60% of saturating (NH4)2SO4concentration. RNA was precipitated by adding (NH4)2SO4 to100% of saturating concentration, then dissolved in RNA ex-traction medium (10 mM Tris-HCl [pH 7.5], 10 mm Mg-acetate,100 mm NaCl, and 10 mm ,3-mercaptoethanol) and desalted bypassage through Sephadex G-25 previously equilibrated withRNA extraction medium. SDS (1%, w/v) was added and thesolution was extracted with an equal volume of phenol (previ-ously equilibrated with RNA extraction medium), and the sep-arated phenol phase was back-extracted with an equal volume ofextraction medium. The aqueous phases were combined and thenucleic acids precipitated by the addition of 2.5 volumes ofabsolute ethanol and storing overnight at -20°C. The precipitatewas collected by centrifugation, dissolved in RNA extractionmedium and extracted several times with equal volumes ofchloroform:isoamyl alcohol (24:1 v/v), until no material wasobserved at the solvent interface. The tRNA-containing fractionwas isolated by the method of Farmerie et al. (10). The aqueousphase was applied to a DEAE-cellulose (Cl) column that hadbeen preequilibrated with RNA extraction medium. The columnwas washed with RNA extraction medium containing 250 mMNaCl until the A260 of the effluent was below 0.05, and then thetRNA-containing material was eluted with RNA extraction me-dium containing 0.7 M NaCl and 1 mM DTT. The collectedRNA was precipitated by the addition of 2.5 volumes of absolute

ethanol. RNA was deacylated by dissolving the precipitate in 500mM Tris-HCl (pH 8.0) and incubating at room temperature for2 h. The deacylated RNA was precipitated by the addition of 0.1volume of 20% (w/v) Na-acetate and 2.5 volumes of absoluteethanol, washed with absolute ethanol, and dried in a vacuumdesiccator. RNA was redissolved in RNA extraction mediumcontaining 1 mM DTT (instead of 10 mm ,B-mercaptoethanol),and stored in small aliquots at -200C.Assay of ALA Formation in Vitro. Assays were carried out as

previously described (35). Enzyme extracts or isolated fractionswere incubated for 30 or 60 min at 300C in 0.25 ml assay buffercontaining 1 mM NADPH, 1 mm Glu, 5 mm ATP, 5 mm K-levulinate, and 1.25 A260 units of RNA prepared as above.Incubation was terminated by the addition of 12.5 ,L of 100%(w/v) TCA, the precipitated protein removed by centrifugation,and 200 uL of the supernatant adjusted to pH 6.8 and reactedwith ethyl acetoacetate to form ALA-pyrrole (20). The productwas quantitated spectrophotometrically at 553 nm after reactionwith an equal volume of Ehrlich-Hg reagent (32).Assay ofGlu-tRNA Formation. A 3H-based filter-binding assay

was adapted from a previously described procedure (18). Incu-bation mixtures contained 1.25 A260 units of RNA obtained asdescribed above, 5 mm ATP, and 100 gM Glu (5 MCi L-[3,4-3H]Glu), in 100 uL of assay buffer. Incubations were carried out for5 min at 30°C, and stopped by the application of 90 ,L samplesto glass microfiber filters, which were immediately immersed in10% (w/v) TCA containing 0.1 M Glu. The filters were washed,dried, and the radioactivity determined as previously described(28).

Preparation of Glu-tRNA for Use as Substrate for ALA For-mation. RNA prepared as described above (2.5 A260 units) wasincubated for 5 min at 300C in 100,ML of assay buffer containingenzyme, 5 mM ATP, and 20 ,uM Glu (5 uCi L-[3,4-3H]Glu). Inmost cases, the added enzyme was 20 ML (312 Mg of protein) ofthe unfractionated enzyme extract obtained by differential pre-cipitation with (NH4)2SO4 at 35 to 60% of saturation. In somecases (where indicated) the added enzyme was 20 ,L (62 MAg ofprotein) of enzyme fraction A-1. The reaction was stopped bythe addition of 200 MuL of termination buffer (0.1 M Mes [pH5.8], 10 mM MgCl2, and 0.1 M Glu). The solution was mixedwith 300 ML of phenol (previously equilibrated with terminationbuffer). The aqueous phase was separated by centrifugation for5 min at 10,OOOg and OC, and extracted with an equal volumeofchloroform:isoamyl alcohol (24: 1, v/v). The nucleic acids wereprecipitated by the addition of 0.1 volume of 20% (w/v) Na-acetate and 2.5 volumes of absolute ethanol. The precipitate wascollected by centrifugation for 30 min at 10,OOOg and 0°C,washed with absolute ethanol, and dried for 1 h in a vacuum

880 AVISSAR AND BEALE Plant Physiol. Vol. 88, 1988

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GLUTAMYL-tRNA IN b-AMINOLEVULINIC ACID BIOSYNTHESIS

dessicator at room temperature. For use as substrate, the precip-itate was dissolved in assay buffer and added to the incubationmixture.

Radioactive Tracer Assay of in Vitro ALA Formation. Incu-bations were carried out for 60 min at 30°C, in 0.25 mL of assaybuffer containing (unless otherwise indicated) the recombinedenzyme fractions, mm NADPH, 5 mM ATP, 5 mM K-levuli-nate, 1.25 A260 units ofRNA and 20 Mm Glu (0.5 MCi L-[3,4-3H]Glu). For the assay of ALA production from L-[3,4-3H]Glu-tRNA or from L-[l-14C]Glu-tRNA, 0.20 mL of assay buffercontaining enzyme fraction A-3, 1 mm NADPH and the Glu-tRNA was incubated at 30°C for 10 min, followed by the additionof enzyme fraction Blu- and further incubation for 10 to 50min. The reaction was terminated by the addition of 25 AL of 1

M citric acid, 250 ML of 10% (w/v) SDS, and 20 ,L of 1 mMALA carrier. The samples were heated at 95°C for 2 min, thenrapidly cooled to room temperature, and applied to 0.25 mLDowex 50W-X8(Na) columns. The columns were washed firstwith 0.5 mL water, then with 0.5 mL of pH 3.0 Na-citratecontaining 50 mM Na+ and 20% (v/v) methanol, then with 1.0mL ofpH 4.25 Na-citrate containing 0.2 M Na+, and finally with0.5 mL water. ALA was eluted with 1.0 mL of pH 6.8 Na-phosphate containing 0.5 M Na+. The eluted ALA was supple-mented with 40 ML ethylacetoacetate and converted to ALA-pyrrole by heating at 95°C for 20 min (20). The solution contain-ing the ALA-pyrrole was cooled, then adjusted to pH 8.5 to 9.0with KOH, and extracted twice with equal volumes of diethylether previously equilibrated with buffer. The aqueous phase wasthen adjusted to pH 2.5 with HCI and extracted three times withequal volumes ofdiethyl ether. The ether extracts were combinedand back extracted with an equal volume of 1 mm HCl, evapo-rated to dryness, and the radioactivity was determined by liquidscintillation spectroscopy in a Beckman LS-1OOC instrument, in5 mL of Econofluor scintillation solution,Chromatography of ALA-Pyrrole. [14C]ALA-pyrrole obtained

as described above was dissolved in diethyl ether and applied toWhatman 3MM paper and chromatographed with n-butanol:n-propanol:5% (w/v) NH40H (2:1:1) (3). The location ofthe ALA-pyrrole was visualized after chromatography by Ehrlich sprayreagent (200 mg of p-dimethylaminobenzaldehyde dissolved in8 mL of ethanol and 2 mL of 12 N HCI) (3). A nonsprayedportion of the chromatograph strip was cut into 1 cm segments,which were immersed into 5.0 mL of Econofluor scintillationfluid and the radioactivity determined by liquid scintillationspectroscopy.Gel Filtration Analysis. A column of Sephadex G-100 (1.5 cm

diameter x 46.5 cm long) was calibrated by determining thepeak elution volumes of several proteins of known mol wt, andthe log mol wt was plotted against the elution volume, yieldinga straight line. The apparent mol wt of an unknown proteincould therefore be determined from its elution volume on thiscolumn.

Native Polyacrylamide Gel Electrophoresis. Samples contain-ing 50 to 120 Mg protein in sample buffer (62 mM Tris-HCl [pH6.8], 10% [v/v] glycerol, 0.7 M ,B-mercaptoethanol, and 0.00 125%[w/v] bromphenol blue) were applied to a 4% (w/v) stacking gel(2 cm, 0.125 M Tris, pH 6.8) overlying a 7.5% (w/v) polyacryl-amide separating gel (12 cm, 0.375 M Tris, pH 8.8), preparedaccording to Laemmli (19), without SDS. The proteins wereseparated by electrophoresis at 30 mamp for 4 h, using Tris-glycine, pH 8.3, electrode buffer at 0°C in a Bio-Rad Protean IISlab Cell. One-half of the gel was fixed and stained, while theother half (containing identical samples) was frozen at -20°Cand the lane containing the sample sliced into 1 mm segments.Groups of 5 to 10 consecutive segments were extracted withassay buffer, and the extracts used for the determination of Glu-tRNA synthetase activity as described above.

Other Procedures. Protein was determined by the dye-bindingmethod of Bradford (5), using BSA as standard. Cell populationdensity was determined with a Coulter Counter (model ZBI,Coulter Electronics).

Materials. L-[3,4-3H]Glu was purchased from Amersham, L-[1-'4C]Glu and Econofluor were from New England Nuclear.DE-23 cellulose was from Whatman, and Sephadex G-25 andG-100 were from Pharmacia. Reactive Blue 2-Sepharose, 2',5'-ADP-agarose, Escherichia coli aminoacyl-tRNA synthetase mix-ture, and mol wt standards for gel filtration and gel electropho-resis were from Sigma. All other chemicals and growth mediumcomponents were from Fisher or Sigma.

RESULTS

Separation of the Enzyme Fractions Required for ALA For-mation from Glu. A typical fractionation procedure, starting with50 g wet weight of cells, yielded 180 mg of enzyme fraction Blu-1, 12 mg of fraction A-1, 1.6 mg of fraction A-2, and 2 mg offraction A-3. Reconstitution ofALA-forming activity after serialaffinity separation required the addition ofenzyme fractions Blu-1, A-1, and A-3 (Table I). Fraction A-2 was not needed, and itappeared to be somewhat inhibitory. Fraction A-2 was omittedfrom all further reconstitutions.Requirement for ALA Formation from Glu. The formation of

ALA from Glu in the reconstituted system was dependent onadded tRNA, Glu, and ATP (omission of any of these compo-nents resulted in less than 5% as much ALA formation as incomplete incubations). K-Levulinate was also required for max-imal ALA accumulation, but in its absence about 60% as muchALA accumulated as in its presence.

In the absence of added NADPH, the amount ofALA formedvaried from 40 to 70% of the amount formed in completeincubations. This apparent cofactor independence resulted fromNADPH carryover along with enzyme fraction A-3, which waseluted from ADP-agarose with 1 mM NADPH. Even thoughfraction A-3 was subsequently passed through Sephadex G-25before use, NADPH was still carried along with the proteinfraction. The NADPH requirement was reestablished by prein-cubating fraction A-3 for 1 h at 0°C, and then passing it throughSephadex G-25 again. After the second gel filtration, only 3% asmuch ALA was formed in the absence of added NADPH as incomplete incubation medium.

Requirements for Glu-tRNA Formation from Glu. Reactionswere carried out in an incubation mixture containing enzymefraction A-1, tRNA, and ATP. Omission of any of these com-ponents from the reaction mixture resulted in lowered levels ofGlu-tRNA recovery (Table II).

Stability of Glu-tRNA in Incubation Buffer. The Glu-tRNAwas degraded at a rapid rate under the incubation conditions,with about half remaining after 10 min (Fig. 2).

Table I. ALA Production by Recombined Enzyme Fractions afterSerial Affinity Chromatography

The complete incubation contained 565 ltg of enzyme fraction Blu-l,1 5 gg of fraction A-1, 21 ug of fraction A-2, 65 ,g of fraction A-3, 1.25A260 units of Chlorella RNA, 1 mm Glu, 5 mM K-levulinate, 5 mM ATP,and I mM NADPH in 250 ML of assay buffer. Incubation was at 30°Cfor 30 min.

Incubation Composition ALA Formation Relative ALA Formationnmol %

Complete 4.92 100-Blu-l 0.13 3-A-1 0.02 0-A-2 7.01 143-A-3 0.04 1

881



4A Synthetase Activity of the Enzyme Fractions. Glu- either substrate (Table IV). When enzyme fraction A-1 wasthetase activity was determined in each of the affinity- omitted from the incubation, incorporation of [3H]Glu into ALAenzyme fractions. Over 70% of the total synthetase was abolished, but incorporation of [3H]Glu-tRNA was at leastcovered was found in fraction A- 1 (Table III). Fraction as efficient as in the completely reconstituted incubations.had the highest specific activity per unit of protein. Requirements for ALA Formation from Glu-tRNA. Formationthe other enzyme fractions also had measurable syn- of labeled ALA from [3H]Glu-tRNA (produced from [3H]Glutivity. and tRNA in a separate incubation with enzyme extract androduction from Glu and Glu-tRNA. Formation of la- isolated by phenol/chloroform extraction) required the presencefrom [3H]Glu or [3H]Glu-tRNA (produced from [3H] of enzyme fractions Blu-l and A-3 (Table V). Omission ofINA in a separate incubation with enzyme extract and NADPH lowered the ALA yield by about 20%. Levulinate wasphenol/chloroform extraction) was measured. Com- not required, and it appeared to inhibit slightly at 5 mM. ATP

onstituted enzyme fractions formed labeled ALA from was not necessary, and addition of 5 mm ATP neither stimulatednor inhibited ALA formation.

Table II. Requirements for Glu-tRNA Formation Chromatography of ALA Produced from Glu-tRNA. The ra-wplete reaction mixture contained, in 100 ML assay buffer, 64 dioactivity recovered in the purified ALA-pyrrole fraction mi-ae fraction A-1, 2.5 A260 units of Chlorella RNA, 5 mm ATP, grated as a single spot on paper chromatograms, and the positionXGlu (5 MCi of L-[3,4-3H]Glu). Incubations were carried out of the peak of radioactivity coincided with that of standard ALA-t 300C. pyrrole used as marker (Fig. 3).cubationRadioactivityRelative Relative Effectiveness of the Glu-tRNA as Substrate for ALA

bposition Incorporated Incorporation Formation. Because enzyme fractions other than A-1 appearedto contain some Glu-tRNA synthetase activity (Table III), the

cpm % conversion of [3H]Glu-tRNA formed by these fractions intonplete 11,200 100 labeled to ALA was examined. Also examined was the transferTP 1,660 15 of label to ALA from [3H]Glu-tRNA formed by a separateLNA 696 6 incubation of Chlorella tRNA with E. coli aminoacyl-tRNA-1 625 6 synthetases. [3H]Glu-tRNA formed by enzyme fraction A-1 ap-incubated 625 6 peared to transfer label to ALA much more efficiently than [3H]

Glu-tRNA formed by the other enzyme fractions or by E. coliaminoacyl-tRNA synthetases (Table VI). The actual efficiency

l1 of label incorporation into ALA was greater than that indicatedby the measured label transfer, because the purification of ALA-pyrrole resulted in a loss of about 35% (data not shown).

Gel Filtration Analysis of Fraction A-1. A l-ml sample ofenzyme fraction A-1 containing 3.2 mg protein, was applied toa Sephadex G-100 column (1.5 cm diameter x 46.5 cm height)equilibrated with assay buffer at 4C and eluted with assay buffer.One-ml fractions were collected and each fraction was assayedfor protein content (A280), Glu-tRNA synthetase activity, andALA forming activity with the reconstituted enzyme systemcontaining enzyme fractions Blu- 1 and A-3. The fractions elutingat the void volume (fraction 30) contained some synthetaseactivity and most of the protein, but were inactive in the recon-stituted ALA forming system. The peak ofALA forming activitycoincided with the second peak of synthetase activity (fractionsI0 20 30 40 35 and 36 in Fig. 4). Based on the elution volumes of standard0 10 20 30 40 50 60 proteins having known mol wt, proteins eluting in these fractions

Time (min)have an apparent mol wt of 74,000 (Fig. 5). An error of ± one

Time (mm) elution fraction corresponds to a mol wt error of ±4,000.Decomposition ofGlu-tRNA under the incubation conditions Native Polyacrylamide Gel Electrophoresis of the FractionL-[3,4-3H]Glu-tRNA produced by fraction A-l was incubated Containing the Synthetase Active in ALA Formation. A 500-,uL00°C in assay buffer. Samples were removed at intervals and portion of fraction 35 from the gel filtration column (above),labeled Glu-tRNA content (100% = 20,800 cpm). containing the peak of the ALA forming activity in the reconsti-

Glu-tRftRNA synseparatedactivity re(

A-1 also IHowever,thetase actALA Pr

beled ALAGlu and tiisolated b~pletely rec

The comrAg of enzynrand 100 Mmfor 5 min al

IncCor

Con-A'-tR-A-Uni

0

FIG. 2. 1

employed. I

for 1 h at 3assayed for

Table III. Aminoacylation ofChlorella RNA by the Various Fractions Obtained in the Serial AffinitySeparation

The reaction mixtures contained, in 100 MAL of assay buffer, 8 ug of the indicated enzyme fraction, 1.25 A260units of Chlorella RNA, 5 mM ATP, and 100 AM Glu (5 MCi of L-[3,4-3H]Glu). Incubations were carried outfor 5 min at 30'C as described in the text.

Enzyme Fraction Activity Specific Activity Total Activity Fraction ofSum-1~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

cpm cpm Mg-' protein cpm %Blu- 1 134 17 2,800,000 16A- 1 8,042 1,005 12,500,000 71A-2 5,128 641 1,000,000 6A-3 5,263 657 1,300,000 7

100

80

60

40

20

01)

0~

882 AVISSAR AND BEALE Plant Physiol. Vol. 88, 1988




Table IV. ALA Production from Glu and Glu-tRNA by ReconstitutedEnzyme Systems

The complete incubations were in 250 ,uL of assay buffer containing565 ,g ofenzyme fraction Blu- 1, 115 gg of fraction A- 1, 65 jig of fractionA-3, 5 mM ATP, 5 mm K-levulinate, 1 mM Glu, and 1 mM NADPH.The substrates were either 0.25 MCi of L-[3,4-3H]Glu combined with 1.25A260 units of RNA, or 142,100 cpm of L-[3,4-3H]Glu-tRNA. [3H]Glu-tRNA was prepared in a separate incubation of [3H]Glu and tRNA withunfractionated enzyme extract and isolation of the product as describedin the text. Incubations were for 60 min at 30°C.

Incubation Radioactive Radioactivity IncorporatedComposition Substrate into ALA

cpmComplete [3H]Glu 1,172Complete [3H]Glu-tRNA 8,133-A-I [3H]Glu 155-A- I [3H]Glu-tRNA 10,271

Table V. Requirements for the Formation ofALA from Glu-tRNAIncubations were carried out in 0.25 mL of assay buffer at 30°C for

10 min following preincubation without enzyme fraction Blu-l for 10min. The complete reaction mixture contained 565 Mg of fraction Blu- 1,65 Mg of fraction A-3, 1 mm NADPH, and Glu-tRNA (133,265 cpmadded to each incubation). [3H]Glu-tRNA was prepared in a separateincubation of [3H]Glu and tRNA with unfractionated enzyme extractand isolation of the product as described in the text.

Composition Radioactivity RelativeIncorporation Radioactivity Incorporated

cpm SComplete 7,123 100-A-3 369 6-Blu-l 351 6-NADPH 5,546 79+ ATP (5 mM) 7,103 100+ Levulinate (5 mM) 6,143 86

U

0

C)~00o

uu -

60 +

40--

20 1-

1 3 5 7 9 1 1 13 15

Migration Distance (cm)

FIG. 3. Paper chromatography of the ALA-pyrrole produced fromGlu-tRNA. '4C-Glu-tRNA was prepared from L-[1-'4C]Glu as describedin the text, and ALA was produced from the '4C-Glu-tRNA in an

incubation containing enzyme fractions Blu-1 (565 Mg) and A-3 (65 Mg).The ALA was derivatized to form ALA-pyrrole, and the purified ALA-pyrrole was chromatographed on Whatman 3MM paper as described inthe text.

tuted assay, was concentrated 10-fold with a Centricon Micro-concentrator having a mol wt cutoff of 30,000, and subjected topolyacrylamide gel electrophoresis under nondenaturing condi-tions. Protein eluted from the gel after electrophoresis was as-sayed for Glu-tRNA synthetase activity (Fig. 6). The majorportion of activity migrated as a single band (Fig. 6). Based onthe migration positions of standard proteins having known molwt, proteins migrating to this region of the gel have an apparentmol wt of 72,000 (Fig. 7). An error of ± one group of gel slicescorresponds to a mol wt error of ±4,000.

DISCUSSION

Although the reaction sequence catalyzing ALA formationfrom Glu in plants and algae has been investigated over a periodof several years, the identity of the biosynthetic intermediateshas not yet been firmly established. The first intermediate in thereaction sequence has been proposed to be a Glu-tRNA on thebasis of several lines of evidence: (a) a tRNA-like molecule wasrequired for ALA formation by all the cell-free extracts testedfrom organisms using this pathway (28); (b) an intact 3'-terminalCCA end on the tRNA was essential for its ability to supportALA formation (29); (c) the formation of Glu-tRNA by activeextracts has been demonstrated (15, 21, 37); and (d) Glu-tRNAserved as a substrate for formation of an intermediate reportedto be Glu-l-semialdehyde in partially fractionated Chlamydo-monas extracts (15). However, because the extract fraction usedin the investigations cited contained both the presumed synthe-tase and dehydrogenase activities, doubt has remained as to theexistence of Glu-tRNA as a free intermediate. Also contributingto the need to further define the role of tRNA are the relativelylow degree of label transfer from Glu-tRNA to the reduced ALAprecursor (15), and the known tRNA requirement for activationof some aminoacyl-tRNA synthetases to form aminoacyl-ade-nylates (25), or to catalyze ATP-PP exchange (8). This leavesopen the possibility that the tRNA requirement in ALA synthesismay result from a role in the formation of some other activatedform of Glu that is the true substrate for the reduction step.Finally, there has been no previous information on the ability ofthe synthetase and dehydrogenase enzymes to independentlycatalyze the partial reactions leading to the reduced ALA precur-sor in the absence of each other.

Recently, the synthetase has been isolated from barley chlo-roplasts and purified by immunoaffinity chromatography (6).This enzyme was reported to be relatively unspecific, amino-acylating with comparable efficiency tRNA of various sources,including E. coli tRNAG"U, and both chloroplast tRNAGlu andtRNAGln (30). This enzyme is assumed to be the only synthetasepresent in chloroplasts, where it activates Glu for both Chl andprotein synthesis. The separation of Chlorella extracts into threerequired enzyme fractions (37) has provided a system withwhich to further characterize the partial reactions and theirrequirements.

In the reconstituted Chlorella system, ALA formation fromGlu required three enzyme fractions, plus tRNA, ATP andNADPH. To demonstrate NADPH dependence, it was necessaryto remove the NADPH that was used to elute enzyme fractionA-3 from the ADP-agarose column, and which remained asso-ciated with the proteins during the subsequent Sephadex G-25filtration. K-Levulinate is required for maximal accumulation ofALA in the complete reconstituted system, but is not requiredin the partial reconstituted system forming ALA from Glu-tRNA. This suggests that ALA dehydratase, the enzyme inhibitedby K-levulinate, may be present in enzyme fraction A-I andabsent from the other fractions or, alternatively, that this enzymeis inactive in partial assay because of the low levels of ALAaccumulating (picomoles as opposed to nanomoles in the com-plete assay).

883



Plant Physiol. Vol. 88, 1988

Table VI. Comparison ofthe Effectiveness in ALA Production ofGlu-tRNA Produced by Aminoacylation ofChlorella RNA with Glu-tRNA Synthetasesfrom Various Sources

For the production of the substrate, incubation mixtures contained, in 100 ,uL of assay buffer, either 62 ,gof enzyme fraction A-1, or a mixture of 73 ,ug of fraction Blu-l, 3 gg of fraction A-2, and 8 sg of fraction A-3, or 450 ,g of E. coli Glu-tRNA synthetase mixture. All incubation mixtures contained additionally 5 mmATP, 2.5 A260 units of Chlorella RNA (prepared from the high-speed supernatant of cell extract), and 20 MmGlu (5 MCi of L-[3,4-3H]Glu). The incubation product was isolated as described in the text. For use in ALAproduction, the isolated incubation product was redissolved in 55 ML of assay buffer, 5 ML were removed fordetermination of radioactivity, and 50 MAL were added to 150 ML of assay buffer containing 65 ,g of fractionA-3, 0.25 ,mol ofNADPH, and 1.25 Mmol of K-levulinate. This mixture was incubated for 10 min, and then565 gg of fraction Blu-l was added (in 50 ML) and the mixture was further incubated for 10 min. Incubationswere terminated and the ALA produced was isolated as described in the text.

Source of Glu-tRNA Radioactivity in Radioactivity Label Transfer fromSynthetase Activity Glu-tRNA in ALA Glu-tRNA to ALA

cpm cpm %A- 1 93,871 33,837 36.0Blu-1 + A-2 + A-3 171,239 4,822 2.8E. coli Glu-tRNA synthetases 7,925 74 0.9

100

a)0L0

(2)a-01)

80

60

40

20

5.00 -

4.75 +3.E0)0-J

O 1

26 30 34 38 42 46

Fraction Number

FIG. 4. Separation of the active constituents in fraction A-1 by gelfiltration. Affinity fraction A-1 (3.2 Mg) was separated on a Sephadex G-100 column into 50 fractions and the fractions were analyzed as describedin the text. Legend: (x), protein content based on A280 (100% = 0.8);(0), Glu-tRNA synthetase activity (100% = 19,200 cpm); (0), activity inthe reconstituted ALA-forming assay (100% = 0.2 A553).

Each of the enzyme fractions obtained has some capacity tocatalyze Glu-tRNA formation. However, only the Glu-tRNAproduced by fraction A- I served as substrate forALA production.This observation explains why fraction A- I is absolutely requiredfor ALA formation from Glu, even though extract lacking frac-tion A-I actively produces Glu-tRNA that is indistinguishablefrom the required species by the aminoacylation assay method.Fraction A-I itself appears to contain at least two synthetaseshaving different apparent mol wt, only one of which is requiredfor ALA formation. Thus, contrary to the situation in barleychloroplasts (6), there are apparently several synthetases capableof catalyzing Glu-tRNA formation in whole-cell Chlorella ex-tracts, but only one of these, that present in the A-I fraction, iscapable of supporting ALA synthesis from Glu. The specificityappears to arise from its ability to aminoacylate a specificallyrequired tRNA that is not charged by the other synthetases (seebelow).The efficiency of label transfer from [3H]Glu-tRNA to ALA

was much higher (nearly 40%) when the [3H]Glu-tRNA substratewas generated by enzyme fraction A-1, compared to substrategenerated by other enzyme fractions, unfractionated enzymeextract, or E. coli aminoacyl-tRNA synthetases. The high effi-

4.50 +-

4.2530 35 40 45 50 55 60 65

Peak Elution Volume (ml)

FIG. 5. Determination of the mol wt of the active component inenzyme fraction A-I by Sephadex G-100 gel filtration. Legend: (0), molwt standards horse heart myoglobin (mol wt = 18,800), bovine erythro-cyte carbonic anhydrase (29,000), ovalbumin (45,000), bovine serumalbumin (66,000), fructose-6-phosphate kinase (84,000), and rabbit mus-cle phosphorylase b (97,400); (0), peak of eluted activity in the reconsti-tuted ALA-forming assay (fraction 35 of Fig. 4).

ciency of label transfer from fraction A-I-generated substrateprobably reflects the specificity of the enzyme in fraction A-Itoward the specific tRNAGIU that is active in the ALA-formingsystem. It was previously shown that several Glu-accepting tRNAspecies are present in the Chlorella tRNA fraction, but only onesubfraction participates in ALA formation (28). The overallfraction of label transferred from [3H]Glu-tRNA to ALA isprobably limited by the low concentration of the [3H]Glu-tRNAand its short half-life (about 10 min) in the incubation buffer.The apparent mol wt of the synthetase active in ALA forma-

tion was determined to be 73,000 ± 5,000 by gel filtration andnative polyacrylamide electrophoresis. This is somewhat lowerthan values of 108,000 and 110,000 reported for the analogoussynthetases from barley chloroplasts (6) and wheat chloroplasts(24, 31), respectively. Both the barley and wheat chloroplastsynthetases were reported to be dimers. By contrast, Glu-tRNAsynthetases of E. coli and Bacillus subtilis are monomers withmol wt values of 56,000 and 65,000, respectively (17, 18, 23).These monomers associate with a 46,000 mol wt protein that is

T T r I

884 AVISSAR AND BEALE




4.000 -

3.000 +

2,000 t

1 .000 4

l-4....-..+-

0

.~~~1~

l~~'i2.1~~

24 6 8 10~~~~~~o-

Migration Distance (cm)

FIG. 6. Glu-tRNA synthetase activity in proteins eluted from a nativepolyacrylamide gel. Native 5% (w/v) polyacrylamide gel electrophoresisof 120 ,ug of protein from fraction 35 of the Sephadex G-100 gel filtrationcolumn (Fig. 4) was followed by the elution of the proteins from the gelslices as described in the text. The eluted samples were assayed for Glu-tRNA synthetase activity as described in the text. The cpm plotted wasobtained from reaction mixtures of 100 uL containing 1.25 A260 unitsRNA obtained as described above, 5 mM ATP, 100 AM Glu (5 ,uCi L-

[3,4-3H]Glu), and 20 gL of eluted protein in assay buffer, treated asdescribed in the text.

5.00 -

4.90 -

4.80 -

4.70 -

4.60 -0

FIG. 7. Det

the peak fractiogel electrophor45,000), bovin(84,000), and r;Glu-tRNA syn

considered toture of the Ciits associaticexcluded.The formal

as cofactor, tisynthetase reregulatory rolfact that isolformation b~ALA from GIof Glu-tRNANow that 1

has been established, it is useful to search for a rationale for thisunusual role for an aminoacyl-tRNA in the biosynthesis of asmall molecule. It is notable that Glu-tRNA formation catalyzedby rat liver Glu-tRNA synthetase apparently does not involveGlu-AMP as an intermediate (8). This conclusion was based onthe observations that the synthetase does not catalyze ATP-PPexchange in the absence of tRNAGIu, and that for the exchangeto occur, the tRNAGIU must contain an intact 3' terminal aden-osine. Moreover, attempts at chemical synthesis of Glu-AMPwere reported to be unsuccessful (4), and the a-hydroxamate ofGlu is extremely unstable (9). It is thus likely that no a-activatedform of Glu other than Glu-tRNA is sufficiently stable to serveas a free intermediate in ALA biosynthesis. The reason for theinstability of other a-activated forms of Glu may be that the e-carboxyl can approach the a region and promote hydrolysis. Itis possible that the approach by the y-carboxyl group to the a

region of Glu-tRNA is sterically prevented by the bulky tRNA.In summary, cell extracts catalyzing ALA-formation from Glu

have been separated into three enzyme fractions and an RNAfraction. These can be reassembled into two groups exhibitingtwo functions: the catalysis of Glu-tRNA formation and theutilization of the Glu-tRNA to form ALA. ATP is needed onlyin the Glu-tRNA-forming step and appears to exert no subse-quent regulatory function, whereas NADPH is needed only inthe second step. It is now established that free Glu-tRNA canserve as an intermediate in the reaction sequence and that thesynthetase and dehydrogenase enzymes are capable of actingseparately and independently.

Acknowledgments-We thank J. D. Weinstein for providing us with the protocolfor the assay of ALA formation from radioactive Glu, and J. G. Ormerod forneSipiui cniicism1II oI 1In manuslnpLE.

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1.2 0.4 0.6 0.8 1.0 mate-tRNA ligase. Carlsberg Res Commun 52: 99-1097. CASTELFRANCO PA, SI BEALE 1983 Chlorophyll biosynthesis: recent advances

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n from the gel filtration column, by native polyacrylamide 1139*esis. Legend: (-), mol wt standards ovalbumin (mol wt = 9. ELLIOT WH, G COLEMAN 1962 A method for studying amino acid activationie serum albumin (66,000), fructose-6-phosphate kinase in crude enzyme preparations. Biochim Biophys Acta 57: 236-244abbit muscle phosphorylase b (97,400); (0), peak of eluted 10. FARMERIE WG, J DELEHANTY, WE BARNErr 1982 Purification of isoaccepting.hetaseactivity.

transfer RNAs from Euglena gracilis chloroplasts. In M Edelman, RB,thetase activity. Hallick, N-H Chua, eds, Methods in Chloroplast Molecular Biology. Elsevier,

Amsterdam, pp 335-346have a regulatory role (18, 23). The subunit struc- 11. FORD SH, HC FRIEDMANN 1979 Formation of 6-aminolevulinic acid from

Worella Glu-tRNA synthetase is not yet known, and glutamic acid by a partially purified enzyme system from wheat leaves.)n with a similar regulatory factor cannot be Biochim Biophys Acta 569: 153-158

i2. FRIEDMANN HC, RK THAUER, SP GOUGH, CG KANNANGARA 1987 5-Ami-nolevulinic acid formation in the archaebacterium Methanobacterium ther-

tion ofALA from Glu-tRNA required only NADPH moautotrophicum requires tRNAG"U. Carlsberg Res Commun 52: 363-371hus establishing the role of ATP as cofactor for the 13. GOUGH SP, CG KANNANGARA 1977 Synthesis of 6-aminolevulinate by a

action only. ATP appears to have no cofactor or chloroplast stroma preparation from greening barley leaves. Carlsberg Res,actinony.ATappars t hav no cfactr or Commun 42: 459-464le in steps subsequent to Glu-tRNA formation. The 14. HAREL E, E NE'EMAN 1983 Alternative routes for the synthesis of 5-amino-ated Glu-tRNA is an efficient substrate for ALA levulinic acid in maize leaves. Plant Physiol 72: 1062-1067y enzyme fractions that are unable to synthesize 15. HUANG D-D, W-Y WANG 1986 Chlorophyll biosynthesis in Chiamydomonaslu indicates that the role oftRNA is in the formation starts with the formation ofglutamyl-tRNA. J Biol Chem 261: 13451-13455L.ratherthanbeing.anallostericactiva

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16. KANNANGARA CG, SP GOUGH, RP OLIVER, SK RASMUSSEN 1984 Biosynthesisrather thzan being an allosterlc activator, of 6-aminolevulinate in greening barley leaves. VI. Activation of glutamate

the true function of Glu-tRNA as an intermediate by ligation to RNA. Carlsberg Res Commun 49: 417-437

E0

0:S

I.

0)

ccC:

z1

zEr

-a

E

0-i

885



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17. KERN D, J LAPOINTE 1979 Glutamyl transfer ribonucleic acid synthetase ofEscherichia coli. Study of the interactions with its substrates. Biochemistry18: 5809-5818

18. KERN D, S POTIER, Y BOULANGER, J LAPOINTE 1979 The monomeric glutamyl-tRNA synthetase of Escherichia coli. Purification and relation between itsstructural and catalytic properties. J Biol Chem 254: 518-524

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20. MAUZERALL D, S GRANICK 1956 The occurrence and determination of -aminolevulinic acid and porphobilinogen in urine. J Biol Chem 219: 435-446

21. MAYER SM, JD WEINSTEIN, SI BEALE 1987 Enzymatic conversion ofglutamateto 6-aminolevulinate in soluble extracts of Euglena gracilis. J Biol Chem262: 12541-12549

22. OH-HAMA T, NJ STOLOWICH, AI SCOTT 1988 5-Aminolevulinic acid formationfrom glutamate via the Cs pathway in Clostridium thermoaceticum. FEBSLett 228: 89-93

23. PROULX M, L DUPLAIN, L LACOSTE, M YAGUCHI, J LAPOINTE 1983 Themonomeric glutamyl-tRNA synthetase from Bacillus subtilis 168 and itsregulatory factor. Their purification, characterization, and the study of theirinteraction. J Biol Chem 258: 753-759

24. RATINAUD MH, JC THOMES, R JULIEN 1983 Glutamyl-tRNA synthetases fromwheat. Isolation and characterization of three dimeric enzymes. Eur JBiochem 135: 471-477

25. RENAUD M, H BACHA, P REMY, J-P EBEL 1981 Conformational activation ofthe yeast phenylalanyl-tRNA synthetase catalytic site induced by tRNAP"Iinteraction: triggering of adenosine or CpCpA trinucleoside diphosphateaminoacylation upon binding of tRNA"ie lacking these residues. Proc NatlAcad Sci USA 78: 1606-1608

26. RIEBLE S, SI BEALE 1988 Enzymatic transformation of glutamate to -amino-levulinic acid by soluble extracts ofSynechocystis sp. 6803 and other oxygenicprokaryotes. J Biol Chem 263: 8864-8871

27. RIEBLE S, JG ORMEROD, SI BEALE 1988 Cell-free extracts from the obligatelyanaerobic photosynthetic bacterium Chlorobium limicola 8327 catalyze con-

ND BEALE Plant Physiol. Vol. 88, 1988

version of glutamate to 6-aminolevulinic acid (abstract 361). Plant Physiol86: S-60

28. SCHNEEGURT MA, SI BEALE 1988 Characterization of the RNA required forbiosynthesis of -aminolevulinic acid from glutamate. Purification by anti-codon-based affinity chromatography and determination that the UUCglutamate anticodon is a general requirement for function in ALA biosyn-thesis. Plant Physiol 86: 497-504

29. SCHON A, G KRuPP, S GOUGH, S BERRY-LOWE, CG KANNANGARA, D SOLL1986 The RNA required in the first step of chlorophyll biosynthesis is achloroplast glutamate tRNA. Nature 322: 281-284

30. SCHON A, D SOLL 1988 tRNA specificity of a mischarging aminoacyl-tRNAsynthetase: glutamyl-tRNA synthetase from barley chloroplasts. FEBS Lett228: 241-244

31. THOMES JC, MH RATINAUD, R JULIEN 1983 Dimeric glutamyl-tRNA synthe-tases from wheat. Kinetic properties and functional structures. Eur J Biochem135: 479-484

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33. WANG W-Y, SP GOUGH, CG KANNANGARA 1981 Biosynthesis of 6-aminolev-ulinate in greening barley leaves IV. Isolation of three soluble enzymesrequired for the conversion ofglutamate to 6-aminolevulinate. Carlsberg ResCommun 46: 243-257

34. WANG W-Y, D-D HUANG, D STACHON, SP GOUGH, CG KANNANGARA 1984Purification, characterization, and fractionation ofthe 6-aminolevulinic acidsynthesizing enzymes from light-grown Chlamydomonas reinhardtii cells.Plant Physiol 74: 569-575

35. WEINSTEIN JD, SI BEALE 1985 Enzymatic conversion of glutamate to 8-aminolevulinate in soluble extracts of the unicellular green alga, Chlorellavulgaris. Arch Biochem Biophys 237: 454-464

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AP



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