biosynthesisof tetrapyrrole pigment precursors1and fractionated by differential (nh4)2so4...

$: Biosynthesisof Tetrapyrrole Pigment Precursors1and fractionated by differential (NH4)2SO4 precipitation at 0°C. Thefraction precipitating at (NH4)2SO4 concentrations between 35 and60%ofsaturation$
Plant Physiol. (1989) 89, 852-8590032-0889/89/89/0852/08/$01 .00/0

Received for publication August 30, 1988and in revised form November 8, 1988

Biosynthesis of Tetrapyrrole Pigment Precursors1

Pyridoxal Requirement of the Aminotransferase Step in the Formation ofb-Aminolevulinate from Glutamate in Extracts of Chiorella vulgaris

Yael J. Avissar and Samuel 1. Beale*Division of Biology and Medicine, Brown University, Providence, Rhode Island 02912

ABSTRACT

The aminotransferase that catalyzes the formation of 6-ami-nolevulinic acid from glutamate-1-semialdehyde or from gluta-mate in a reconstituted enzyme system was isolated and partiallypurified from Chlorella vulgaris. The apparent molecular weightof the aminotransferase was determined by Sephadex G-100 andUltrogel AcA 54 gel filtration to be 60,000 ± 5,000. Catalyticactivity of the aminotransferase required pyrixodal phosphate(PALP). The cofactor could not be removed by gel filtration afterexposure of the enzyme to PALP. Aminotransferase was inhibitedby gabaculine (3-amino-2,3-dihydrobenzoic acid). The concentra-tion of gabaculine required for half maximal inhibition was about0.05 micromolar. Aminotransferase activity could be regainedupon the removal of gabaculine by gel filtration and supplement-ing the assay medium with PALP. Neither the inhibitory action ofgabaculine nor its reversibility was affected by preincubation ofthe enzyme with the keto acids levulinate and &-aminolevulinicacid.

the reaction to inhibitors that are considered to be pyridoxalantagonists, such as gabaculine and aminooxyacetate (5, 10,13, 21). However, previous attempts to demonstrate directlya dependence of enzyme activity on PALP were unsuccessful(6, 20). The apparent lack of dependence of the aminotrans-ferase on PALP was explained as resulting from the existenceof a tightly bound pyridoxal or pyridoxamine phosphate thatis not removed from the enzyme by the various purificationprocedures employed during the preparation of the enzymeextract (5).We now report the direct demonstration of the dependence

of aminotransferase activity in vitro on added PALPand the positive effect ofPALP on the recovery ofaminotrans-ferase activity following preincubation of the enzyme withgabaculine.

MATERIALS AND METHODS

The transformation ofglutamate to the tetrapyrrole precur-

sor ALA2 by the five-carbon pathway in plants, algae, andsome bacteria involves the participation ofthree enzymes anda specific tRNA molecule (Fig. 1). The characteristics oftheseenzymes and the tRNA are remarkably similar in the variousorganisms and in the cell-free preparations obtained fromthem (2). The terminal step of the reaction sequence, cata-lyzed by an aminotransferase, is still poorly characterizedsince there is yet no agreement among the investigators aboutthe identity ofthe biological substrate ofthe aminotransferase,the catalytic mechanism (1 1), and the cofactor requirementof the reaction (5).An enzyme capable of catalyzing the transformation of

chemically synthesized GSA to ALA was purified from ex-

tracts of barley chloroplasts (6, 19) and from the green algaChlamydomonas (20). These preparations catalyzed ALA for-mation from GSA in the absence of any other substrate or

added cofactor. The involvement of PALP as a cofactor forthe aminotransferase step was suggested by the sensitivity of

' Supported by National Science Foundation grant DMB85-18580.2 Abbreviations: ALA, 6-aminolevulinic acid; gabaculine, 3-amino-

2,3-dihydrobenzoic acid; GSA, glutamate-1 -semialdehyde; PALP,pyridoxal phosphate; BB and BU, Blue-Sepharose bound and un-

bound fractions, respectively.

Growth of Algae

Chlorella vulgaris Beijerinck strain C-10 was grown in batchculture in a glucose-based liquid medium as previously de-scribed (21) on rotary shakers at 25°C. Strain C-10 forms Chlonly in the light. Cells were grown in complete darkness untillate exponential growth phase and were transferred to thelight (32 ,uE m-2s-', 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 (22), resus-pended in homogenization buffer (0.1 M Tricine [pH 7.9], 0.3M glycerol, 15 mM MgCl2, 1 mM DTT, 2 mM EDTA, and 2.5ppm PMSF), and broken by passage through a French pres-sure cell at 23,000 p.s.i. The homogenate was centrifuged atl0,OOOg for 10 minutes to remove cell debris and unbrokencells, stirred with 0.5 M NaCl on ice for 20 min, and thencentrifuged at 264,000g at 2°C for 90 min. The clear upperportion of the supernatant was strained through glass wooland fractionated by differential (NH4)2SO4 precipitation at0°C. The fraction precipitating at (NH4)2SO4 concentrationsbetween 35 and 60% of saturation was harvested by centrif-ugation, redissolved in assay buffer (50 mM Tricine [pH 7.9],1 M glycerol, 15 mM MgCl2, and 1 mM DTT), and desaltedby passage through Sephadex G-25 that had been preequili-

852

https://plantphysiol.orgDownloaded on April 19, 2021. - Published by Copyright (c) 2020 American Society of Plant Biologists. All rights reserved.

https://plantphysiol.org

PYRIDOXAL PHOSPHATE IN b-AMINOLEVULINIC ACID BIOSYNTHESIS

COOH COOH COOH COOH

CH2 CH2 CH2 CH2I tRNAGI1, ATP, Mg 2+ NADPH _ HI PALP

Glutamyl-tRNA Synthetase I Dehydrogenase IH2 AminotransferaseCHNH2 CHNH2 CHNH2 C=O

COOH CO CHO CH2NH2

tRNAGIU

Glutamic Acid Glutamyl-tRNA GSA ALA

Figure 1. Proposed biosynthetic sequence of ALA formation from glutamate via the RNA-dependent five-carbon pathway. The cofactorrequirements, enzyme activities, and intermediate products are illustrated. GSA, the reduced intermediate illustrated, is one of several structuresproposed for the immediate precursor of ALA.

brated with assay buffer. Fractionation of this enzyme extracton Reactive Blue 2-Sepharose was carried out as previouslydescribed (22). The unbound fraction (BU) and the fractioneluted with 0.5 M NaCl (BB) were precipitated by addition of(NH4)2SO4 to 70% of saturating concentration, the precipi-tates were redissolved in assay buffer, desalted by passagethrough Sephadex G-25, and stored in aliquots at -75°C.

RNA Extraction and Partial Purification

RNA was prepared as previously described (16). Sources ofthe RNA were the lower, pigmented portiQn ofthe high-speedsupernatant remaining after the removal of the clear upperportion for enzyme fractionation, and the supernatant frac-tion from the precipitation of the enzymes at 60% of saturat-ing (NH4)2SO4 concentration. The tRNA-containing fractionwas isolated by the method ofFarmerie et al. (4). The aqueousphase was applied to a DEAE-cellulose (C 1) column that waspreequilibrated with RNA extraction medium (10 mM Tris-HCI [pH 7.5], 10 mm Mg-(acetate)2, 10 mM ,f-mercaptoetha-nol), the column was washed with RNA extraction mediumcontaining 250 mM NaCl until the A260 of the effluent wasbelow 0.05, and then the tRNA-containing material waseluted with RNA extraction medium containing 0.7 M NaCland 1 mM DTT instead of 10 mM ,B-mercaptoethanol. TheRNA was precipitated by the addition of 2.5 volumes ofabsolute ethanol. RNA was deacylated by dissolving the pre-cipitate in 500 mM Tris-HCl (pH 8.0) and incubating at roomtemperature for 2 h. The deacylated RNA was precipitatedby the addition of 0.1 volume of 20% (w/v) Na-acetate and2.5 volumes of absolute ethanol, washed with absoluteethanol, and dried in a vacuum desiccator. RNA was redis-solved in RNA extraction medium containing 1 mm DTTinstead of 10 mm f3-mercaptoethanol, and stored in smallaliquots at -20°C.

Assays of ALA Formation in Vitro

Assays ofALA formation from glutamate were carried outin a reconstituted enzyme system as previously described (21).Enzyme extracts or isolated fractions were incubated for 30min at 30°C in assay buffer containing 1 mM NADPH, 1 mM

glutamate, 5 mM ATP, 5 mM K-levulinate, and 1.25 or 2.50A260 units of RNA prepared as above in a final volume of0.25 mL.ALA formation from GSA was assayed following the pro-

cedure of Hoober et al. (5) using a GSA preparation (5, 10).Enzyme fraction BU was incubated for 20 or 30 min at 30°Cin 0.25 mL of assay buffer containing 5 mM levulinate and0.1 mg ofGSA preparation.

Incubations were terminated by the addition of 12.5 ML of100% (w/v) TCA, the precipitated protein was removed bycentrifugation, and 200 ML of the supernatant was adjustedto pH 6.8 and reacted with ethyl acetoacetate to form 1-methyl-2-carboxyethyl-3-propionic acid pyrrole (12). Theproduct was quantitated spectrophotometrically at 553 nmafter reaction with an equal volume of Ehrlich-Hg reagent(18).

Separation of Enzyme from Preincubation Medium

Enzyme samples (0.2 mL) that were preincubated undervarious conditions as indicated were applied to a column ofSephadex G-25 (5 cm long x 0.5 cm diameter), previouslyequilibrated with assay buffer, and eluted with assay buffer at4°C.

Estimation of the Mol Wt of the Aminotransferase by GelFiltration

Blue-Sepharose fraction BU obtained as described above,containing 110 mg of protein in 50 mL of column buffer(assay buffer supplemented with 20 uM PALP), was appliedto a DEAE-cellulose column (10.2 cm long x 2.5 cm diame-ter) previously equilibrated with column buffer. The proteinswere eluted with a linear gradient ofNaCl (0-0.5 M) in columnbuffer. The fractions containing aminotransferase activity(peak activity eluted at 0.28 M NaCl) were pooled and theprotein was collected by centrifugation, dissolved in columnbuffer, and desalted by gel filtration through Sephadex G-25.The desalted sample, containing 20 mg of protein in 4 mL ofcolumn buffer, was applied to an Ultrogel AcA 54 column(90 cm long x 2.7 cm diameter) and eluted with columnbuffer at the rate of 21 mL/h at 5°C. The A280 of the effluent

853



Plant Physiol. Vol. 89, 1989

stream was continuously monitored. Effluent fractions (5.25mL) were assayed for aminotransferase activity in the recon-stituted enzyme system described above. Alternatively, theDEAE-cellulose-purified protein was applied to a SephadexG-100 column (62 cm long x 1.5 cm diameter) directly afterredissolving the (NH4)2SO4 pellet. One mL of assay buffercontaining 30 mg of protein was applied, and the column waseluted with assay buffer at the rate of 18 mL/h at 8°C. Two-mL fractions were collected and assayed for aminotransferaseactivity. Both columns used for mol wt determinations werecalibrated with several proteins of known mol wt.

Other Procedures

Protein was determined by the dye-binding method ofBradford (3) or by measuring A280 of the effluent fractions.Cell population densities were determined with a CoulterCounter (model ZBI, Coulter Electronics). The concentrationofGSA was estimated by the modified 3-methyl-2-benzothia-zolone assay (10, 15).

Chemicals

GSA was a generous gift from C. G. Kannangara (CarlsbergLaboratory, Copenhagen, Denmark). Cellulose DE-23 waspurchased from Whatman, Ultrogel AcA 54 from LKB, andgabaculine from Fluka. Sephadex G-100, protein mol wtmarkers, and Reactive Blue 2-Sepharose were from Sigma,and all other chemicals from Sigma or Fisher.

RESULTS

Requirements for ALA Formation from Glutamate andGSA

ALA formation from glutamate required reconstitution ofBlue-Sepharose fractions BB and BU and occurred in thepresence ofglutamate, ATP, Mg2", and NADPH as previouslyreported (1, 22). ALA formation from GSA required onlyfraction BU, but also occurred in the presence ofthe completereconstituted system plus cofactors. Fraction BB did notcatalyze ALA formation from GSA, either in the presence orabsence of cofactors. The ability of fraction BU to catalyzeALA formation from GSA was lost by heating at 98°C for 2min.The quantity of GSA preparation added to incubation

mixtures (0.1 mg) contained approximately 16 nmol ofGSAwhen measured by the colorimetric assay (10, 15), correspond-ing to 2.1% GSA content (mol wt = 131.1), similar to thereported value (5). When the aminotransferase reaction con-taining 0.1 mg of GSA preparation was allowed to run tocompletion, the yield of ALA was 15.9 nmol, close to thetheoretical yield.

Stimulation of ALA Formation from Glutamate by PALP

ALA formation from glutamate was assayed in the recon-stituted enzyme system in incubations containing a constantamount of Blue-Sepharose fraction BB and various amountsof fraction BU (Fig. 2). The amount of fraction BU in theincubation is expressed as a percentage of the ratio in which

3.6

-

0E

,o

0

IL

-J

2.7

1.8

0.9

0.00 50 100 1 50

Amount of Fraction BU (%)

Figure 2. Stimulation of ALA formation from glutamate by PALP inthe presence of various amounts of enzyme fraction BU and aconstant amount of fraction BB. The amount of fraction BU is ex-pressed as a percentage representing the ratio of the two fractionsobtained from the same amount of cell extract. Reconstituted enzymeassay in 250 ML of assay buffer contained 0.67 mg of fraction BBprotein, 0.17-2.03 mg of fraction BU protein, 1.25 A260 units ofChlorella tRNA, 1 mm NADPH, 1 mm glutamate, 5 mm K-levulinate,and 5 mm ATP. Incubation was at 300C for 30 min in the presence(@) or absence (0) of 20 Mm PALP.

the two fractions are obtained from cell extract (100% = 1.35mg ofBU and 0.67 mg ofBB protein). The degree ofdepend-ence of ALA production on added PALP was a function ofthe relative proportions of enzyme fractions BB and BU inthe incubation. A marked dependence on added PALP wasobserved only when the relative amount of fraction BU waslow. The PALP dependence decreased with increasing relativeamount of fraction BU. There was an apparent tendency ofthe two curves to converge at even higher amounts of fractionBU relative to fraction BB present in the incubation.

Effect of PALP Concentration on ALA Production fromGlutamate and from GSA

Addition of PALP to a reconstituted enzyme system con-taining 0.17 mg offraction BU protein and 0.67 mg offractionBB protein resulted in a significant increase in ALA produc-tion from glutamate (Fig. 3). ALA production from GSA byfraction BU alone was also strongly stimulated by PALP.Significant stimulation was observed at PALP concentrationsabove 0.1 Mm, and maximal stimulation occurred at approxi-mately 3 Mm. Very little additional stimulation was observedat 20 Mm PALP. Nonenzymatic ALA production from GSAwas unaffected by PALP within the concentration rangetested.

Effect of Preincubation of Fraction BU with PALP

Preincubation of fraction BU with PALP for 2 h on iceresulted in a 5.4-fold increase in activity when assayed in theabsence of added PALP (Table I). The enzyme that waspreincubated with PALP retained its high activity and ac-

854 AVISSAR AND BEALE



PYRIDOXAL PHOSPHATE IN b-AMINOLEVULINIC ACID BIOSYNTHESIS

1 2

0Ec

c0

0IL

-j

9

6

3

O0 1 .

0.0 0.5 1.0 1.5 2.0 2.5 3.0

PALP Concentration (gM)Figure 3. Effect of PALP concentration on ALA formation in thereconstituted enzyme assay with glutamate as substrate (0) or withfraction BU alone with GSA as substrate (0). The values at 20 gMPALP are shown against the right border. Reconstituted enzymeassay in 250 MuL of assay buffer contained 0.67 mg of fraction BBprotein, 0.17 mg of fraction BU protein, 2.5 A2w units of ChlorellatRNA, 1 mm NADPH, 1 mm glutamate, 5 mm K-levulinate, and 5 mMATP. Incubation was at 300C for 30 min. Assay with GSA as substratecontained (in 250 ML of assay buffer) 0.17 mg of fraction BU protein,0.1 mg of GSA preparation, and 5 mm K-levulinate. Nonenzymic ALAformation from GSA was assayed in parallel at the various PALPconcentrations and was found to be 1.34 nmol ALA produced per 30min. The values presented have not been corrected for this back-ground ALA formation.

Table I. Effect of Preincubation with PALP on ALA FormationThe complete incubation in 250 ML assay buffer contained 0.17

mg of fraction BU protein (preincubated on ice with 20 Mm PALP for2 h and subsequently gel filtered through Sephadex G-25, whereindicated), 0.67 mg of fraction BB protein, 2.5 A2w units of ChlorellatRNA, 1 mm NADPH, 1 mm glutamate, 5 mm K-levulinate, and 5 mMATP. Incubation was at 300C for 30 min in the presence or absenceof added 20 Mm PALP.

Added PALP Added PALP ALA Formationin Preincubation in Assay

nmol %

- - 1.31 100+ 7.14 545

+ - 7.14 545+ + 7.12 543

quired independence of additional PALP even after removalof unbound PALP by gel filtration through Sephadex G-25prior to incubation.

Inhibition of ALA Formation by Gabaculine

Gabaculine inhibited ALA formation both from glutamatein the reconstituted ALA-forming system and from GSA byenzyme fraction BU (Table II). In the absence ofadded PALP,the degree of inhibition of ALA formation from GSA in-creased with increasing gabaculine concentration, half-maxi-

Table II. Effect of Gabaculine Concentration on ALA Formation fromGlutamate or GSA in the Presence or Absence of Added PALPEnzyme fractions BU and BB were prepared in the presence of

PALP and freed of unbound PALP by Sephadex G-25 gel filtrationbefore use. Enzyme fraction BU (0.17 mg of protein, for ALA synthesisfrom GSA) or reconstituted enzymes (0.17 mg of fraction BU proteinand 0.67 mg of fraction BB protein, for ALA synthesis from glutamate)were preincubated for 20 min at 300C in assay buffer containing 20AM PALP and/or the indicated concentration of gabaculine. This wasfollowed by addition the appropriate substrates and cofactors forALA synthesis from GSA or glutamate and incubation at 30°C for anadditional 25 min. Final incubation volumes were 250 AL. For ALAsynthesis from GSA, incubation mixtures contained 0.1 mg of GSApreparation, 5 mm K-levulinate, and the preincubation ingredients.For ALA formation from glutamate, incubation mixture contained 1.25Am units of Chlorella tRNA, 1 mM NADPH, 1 mm glutamate, 5 mMATP, 5 mm K-levulinate, and the preincubation ingredients.

PreincubationConditions ALA Relative

Substrate FormaLA ALAAdded Added Formation ForrationPALP gabaculineMM MM nmol %

GSA 20 0.00 8.3 10020 0.05 6.3 7620 0.10 1.3 160 0.00 6.8 820 0.05 4.1 490 0.10 0.7 8

Glutamate 20 0.00 3.9 10020 0.05 3.1 7920 0.10 1.0 260 0.00 3.9 1000 0.05 1.6 410 0.10 0.2 5

mal inhibition occurred at approximately 0.05 Mm gabaculine,and inhibition was nearly complete at 0.1 uMm gabaculine (Fig.4). In the absence of added PALP, half-maximal inhibition ofALA formation from glutamate also occurred at approxi-mately 0.05 Mm gabaculine. Inhibition ofALA formation fromeither glutamate or GSA occurred at slightly higher gabaculineconcentration in the presence of 20 gM added PALP(Table II).To test the reversibility of the gabaculine inhibition, sam-

ples containing fraction BU were preincubated in assay bufferwith 5 uM gabaculine and/or 20 gM PALP. ALA formationfrom glutamate was then assayed in the reconstituted enzymesystem containing the preincubated fraction BU and assaymedium containing 20 Mm PALP (Table III). Gel filtration offraction BU following the preincubation with gabaculine re-stored ALA-forming activity in the reconstituted system.Reactivation occurred upon gel filtration whether or notPALP was present during the preincubation. In samples thatwere not gel filtered after preincubation, the presence of 20,aM PALP during the preincubation with gabaculine slightlyattenuated the inhibition of ALA formation.

Effects of Keto Acids on Inhibition of AminotransferaseActivity by GabaculineSamples of fraction BU were preincubated with 5 mM

levulinate or ALA for 30 min at 30°C prior to the addition of

855




1 00

75000

500

0

O-2

0 20 40 60 80 100

Gabaculine Concentration (nM)

Figure 4. Inhibition of ALA formation from GSA by gabaculine.Enzyme fraction BU (0.17 mg of protein) was preincubated for 20min at 300C in assay buffer containing the indicated concentration ofgabaculine. This was followed by addition the appropriate substratesand cofactors for ALA synthesis from GSA (see text) and incubationat 300C for an additional 20 min. Final incubation volumes were 250ML. ALA formed in the control incubation, without added gabaculine,was 7.66 nmol.

Table Ill. Reversibility of Gabaculine Inhibition of ALA FormationThe complete incubation in 250 MgL of assay buffer contained 0.17

mg of fraction BU protein (preincubated on ice with 5 AM gabaculine,20 uM PALP, or assay buffer for 1 h and subsequently gel filteredthrough Sephadex G-25, where indicated), 0.67 mg of fraction BBprotein, 1.25 A2w units of Chlorel/a tRNA, 1 mm NADPH, 1 mMglutamate, 5 mm K-levulinate, and 5 mm ATP. Incubation was at 300Cfor 30 min in the presence of 20 Mm PALP.

Preincubation Gel Filtration after ALAConditions Preincubation Formation

nmol %

Assay buffer - 5.52 100Gabaculine - 0.10 2Gabaculine + 5.42 98Gabaculine + PALP - 0.40 7Gabaculine + PALP + 5.28 96

gabaculine, then, after an additional preincubation for 1 h at0°C, the samples were gel filtered through Sephadex G-25 andassayed for ALA formation in the reconstituted enzyme sys-tem. The presence of ALA or levulinate during the preincu-bation had little influence on the effect of preincubation withgabaculine on the final activity of the reconstituted system(Table IV). In all cases, preincubation with gabaculine greatlylowered ALA-forming activity in assays without added PALP,and activity was largely restored by addition of PALP to theassay medium. The BU fraction used in this experiment hadbeen previously exposed to PALP and was, therefore, initiallyunaffected by added PALP. Even after the various preincu-bations the activity (ofthe samples not exposed to gabaculine)was only slightly stimulated by added PALP.

Table IV. Effects of Keto Acids on Gabaculine Inhibition of ALAFormation

The complete incubation in 250 ML of assay buffer contained 0.37mg of fraction BU protein, 0.67 mg of fraction BB protein, 1.25 A260units of Chlorella tRNA, 1 mm NADPH, 1 mm glutamate, 5 mm K-levulinate, and 5 mm ATP. Incubation was at 30°C for 30 min in thepresence or absence of 20 AM PALP. Prior to the incubations, fractionBU was incubated with 20 uM PALP and then gel filtered throughSephadex G-25 to remove unbound PALP. Portions were then prein-cubated with the indicated keto acid (5 mM), gel filtered again toremove the keto acid, then preincubated with 5 Mm gabaculine whereindicated, and gel filtered once again to remove unbound gabaculine.

Keto Acid in Gabaculine in Assay without Assay withFirst Second Added PALP Added PALP

Preincubation Preincubation

nmol ALA % nmol ALA %- - 5.60 100 6.10 100- + 0.15 3 4.73 78

K-levulinate + 0.05 1 3.92 64ALA + 0.51 9 4.03 66

00

N

0OD

CY

0

0

a.

0.06

co0.04 ~

0

0-

0.02 <

0.00165 205 245 285 325

Elution Volume (ml)

Figure 5. ALA-forming activity (0) and protein content (0) of fractionseluted from the Ultrogel AcA 54 column. Protein content was deter-mined by measuring the A260 of the effluent stream. A280values above2.0 are indicated against the upper border. ALA production wasassayed in the reconstituted enzyme system (in 250 ML assay buffer)containing 0.1 mL of column eluate, 0.67 mg of fraction BB protein,1.25 Am units of Chlorella tRNA, 1 mm NADPH, 1 mm glutamate, 5mM K-levulinate, and 5 mm ATP. Incubation was at 300C for 30 min.

Determination of the Mol Wt of the Aminotransferase byGel Filtration

Fraction BU was chromatographed on a calibrated columnof Ultrogel AcA 54. The effluent stream was continuouslymonitored for A280 and effluent fractions were assayed foraminotransferase activity in the reconstituted enzyme system.The apparent mol wt ofthe enzyme deduced from the locationof the peak of enzyme activity is 59,200 (Figs. 5 and 6). Adeviation of +one 5.25-mL effluent fraction corresponds to amol wt deviation of±3,000. A sample ofthe same preparationwas also gel filtered through a calibrated column of SephadexG-100, and yielded a mol wt value of 61,600 (Fig. 7). A

856 AVISSAR AND BEALE



PYRIDOXAL PHOSPHATE IN 6-AMINOLEVULINIC ACID BIOSYNTHESIS

5.1

4.8

4.6-

4.4 4

4.2t

4.0

2210O 260 300 340 380 420

Peak Elution Volume (ml)

Figure 6. Determination of the mol wt of the aminotransferase activ-ity in enzyme fraction BU by Ultrogel AcA 54 gel filtration. Legend:(@) mol w(1 8,800), c(66,000); (Ituted ALA

5.0

4.8

E 4.6-E

4.4

4.2

Figure 7.ity in enzy(@) mol wbovine eryBSA (66,0'cle phosplactivity in

deviationmol wt dltwo deter

The oN

contain Prole of P.been bas4

0

11

0 CH2HO%j* I

.-C\ CH2H \/

C

H NH2

GSA (Cyclized)

8 H1O c

H2C I

,C

CH11CH

H NH2

GabaculineFigure 8. Proposed cyclic structure for the substrate of the ALA-forming aminotransferase (PM Jordan, RP Sharma, MJ Warren, per-

sonal communication) and the structure of gabaculine.

t standards cytochrome c (mol wt = 12,400), myoglobin aminotransferase and on the observed inhibition of ALA-marbonic anhydrase (29,000) ovalbumin (45,000), and BSA forming activity by PALP antagonists, aminooxyacetate andD) peak of eluted aminotransferase activity in the reconsti- especially gabaculine (5, 17). However, until now there has,-forming assay. been no direct evidence of a requirement for PALP in any of

the in vitro ALA-forming systems tested. By adopting a prep-

aration procedure for the enzyme that excludes PALP fromthe buffers at all stages, it has now been possible to demon-strate a PALP requirement of the aminotransferase reaction.Enzyme extract prepared from greening Chlorella cells in theabsence of PALP has a strong dependence on added PALPfor maximal activity. Once the enzyme is exposed to thecofactor, the binding is so tight that subsequent gel filtrationdoes not remove it, and enzyme activity becomes independentof added PALP thereafter. Previous attempts to demonstratePALP dependence of the Chlorella ALA-forming systemfailed because the buffers used for cell breakage and initialpurification steps contained 20,uM PALP (22), and efforts tosubsequently remove the PALP by gel filtration areineffective.

The degree of dependence on PALP is a function of theconcentration of aminotransferase relative to the other en-

Peak Elution Volume (ml) zymes in the reconstituted assay system. This enzyme concen-

Determination of the mol wt of the aminotransferase activ- tration effect on the PALP dependence probably results fromFme fraction BU by Sephadex G-1 00 gel filtration. Legend: a small portion of aminotransferase molecules that carry/t standards horse heart myoglobin (mol wt = 18,800), bound PALP even in enzyme extracts that have been preparedrthrocyte carbonic anhydrase (29,000), ovalbumin (45,000), in the absence of added PALP. In reconstitutions containing00), fructose-6-phosphate kinase (84,000), and rabbit mus- the aminotransferase enzyme in great excess, relative to thehorylase b (97,400); (0) peak of eluted aminotransferase other required enzymes, the PALP-containing portion of the

the reconstituted ALA-forming assay. aminotransferase molecules is sufficient to catalyze ALA for-

mation at nonlimiting rates.

of ±one 2-mL effluent fraction corresponds to a Chlorella aminotransferase is extremely sensitive to gaba-eviation of ±6,500. The mean mol wt value for these culine. The gabaculine concentration required for half-maxi-rminations is 60,400 ± 5,000. mal inhibition of ALA formation from GSA or glutamate,

0.05 ,M, is less than one tenth of the reported KI for inhibitionDISCUSSION of y-aminobutyric acid:a-ketoglutaric acid transaminase (13,

14). The extreme sensitivity of aminotransferase to gabaculineverwhelming majority of known aminotransferases may result from a very close structural resemblance of the'ALP as the prosthetic group. The presumed cofactor inhibitor to the normal substrate, for which a cyclic structure

'ALP in the formation of ALA from glutamate has has recently been proposed (PM Jordan, RP Sharma, MJed on the hypothetical mechanism of action of the Warren, personal communication). The cyclic compound, 3-

nl

857

f%.lu




amino-2-hydroxy-tetrahydropyran-1-one, is formally an in-ternal ester between the y-carboxyl group and a hydroxylgroup of hydrated GSA. The proposed cyclic substrate bearsa strong resemblance to gabaculine (Fig. 8).

Following incubation with gabaculine, aminotransferaseactivity was restored upon removal of the inhibitor by gelfiltration and addition of PALP. The mechanism describedfor gabaculine inhibition of y-aminobutyric acid:a-ketoglu-taric acid transaminase involves covalent and irreversiblemodification of the PALP cofactor and formation of anadduct that is tightly bound to the enzyme (13). The adductitself is not covalently attached to the enzyme and, dependingon the particular enzyme, might be subject to dissociation ordisplacement from the enzyme by PALP. In the case of theaminotransferase involved in ALA formation, the restorationof gabaculine-inactivated enzyme after removal of gabaculineby gel filtration and addition PALP indicates that, as with 'y-aminobutyric acid:a-ketoglutaric acid transaminase, gabacu-line interacts with the enzyme-bound PALP cofactor ratherthan with the enzyme itself. The observation that gabaculine-treated enzyme is reactivated by gel filtration followed bysupplementation with PALP, whereas unreacted enzyme ap-pears to retain its PALP through gel filtration, indicates thatthe gabaculine-PALP adduct is less tightly bound to theenzyme than unreacted PALP, or that, after gel filtration, inthe absence of excess free gabaculine, the enzyme-boundgabaculine-PALP adduct is readily displaced by PALP.We have attempted to reproduce the enhancement by keto

acids of the gabaculine inhibition that was reported for thebarley enzyme (5). No significant effect on gabaculine inhi-bition or its reversibility was detected in the Chlorella systemin the presence of either of the two keto acids, levulinic acidand ALA.

Accurate determination of the mol wt of the aminotrans-ferase enzyme has been difficult because the enzyme activityis normally present in cell-free extracts in large excess of theother enzyme activities required for ALA formation fromglutamate, thus it is difficult to make the aminotransferaserate limiting in reconstituted assays. The barley enzyme wasvariously reported to have a mol wt of 67,000 (7) and 80,000(8, 9). The Chlamydomonas enzyme was reported to have anapparent mol wt of 45,000 (20) and the Chlorella enzyme waspreviously reported to have an apparent mol wt of 67,000(22). The present results obtained with two different gelfiltration materials indicate a somewhat lower value60,000 ± 5,000.

In summary, Chlorella aminotransferase was found to bedependent on PALP for maximal activity. The enzyme bindsPALP tightly. Activity is sensitively, but reversibly inhibitedby gabaculine, and the gabaculine inhibition is unaffected bypreincubation of the enzyme preparation with the keto acidsALA and levulinate. The apparent native mol wt of Chlorellaaminotransferase is 60,000.

ACKNOWLEDGMENTS

We thank C. G. Kannangara for supplying the sample of GSAused in our experiments, P. M. Jordan for making data available tous before publication, S. M. Mayer and A. H. Mehler for critical

comments on the manuscript, and J. D. Weinstein for helpful discus-sions.

LITERATURE CITED

1. Avissar YJ, Beale SI (1988) Biosynthesis oftetrapyrrole pigmentprecursors: Formation and utilization ofglutamyl-tRNA for 6-aminolevulinic acid synthesis by isolated enzyme fractionsfrom Chlorella vulgaris. Plant Physiol 88: 879-886

2. Beale SI, Weinstein JD (1989) Tetrapyrrole metabolism in pho-tosynthetic organisms. In HA Dailey, ed, Biosynthesis of Te-trapyrroles. Macmillan, New York (in press)

3. Bradford MM (1976) A rapid and sensitive method for thequantitation of microgram quantities of protein utilizing theprinciple of protein-dye binding. Anal Biochem 72: 248-254

4. Farmerie WG, Delehanty J, Barnett WE (1982) Purification ofisoaccepting transfer RNAs from Euglena gracilis chloroplasts.In M Edelman, RB Hallick, N-H Chua, eds, Methods inChloroplast Molecular Biology. Elsevier, Amsterdam, pp 335-346

5. Hoober JK, Kahn A, Ash DE, Gough S, Kannangara CG (1988)Biosynthesis of 6-aminolevulinate in greening barley leaves.IX. Structure of the substrate, mode of gabaculine inhibition,and the catalytic mechanism of glutamate 1-semialdehydeaminotransferase. Carlsberg Res Commun 53: 11-25

6. Kannangara CC, Gough SP (1978) Biosynthesis of 6-aminolevu-linate in greening barley leaves: glutamate I-semialdehydeaminotransferase. Carlsberg Res Commun 43: 185-194

7. Kannangara CG, Gough SP (1979) Biosynthesis of 6-aminolevu-linate in greening barley leaves. II. Induction of enzyme syn-thesis by light. Carlsberg Res Commun 44: 11-20

8. Kannangara CG, Gough SP, Bruyant P, Hoober JK, Kahn A,von Wettstein D (1988) tRNAGIU as a cofactor in 6-aminolev-ulinate biosynthesis: steps that regulate chlorophyll synthesis.Trends Biochem Sci 13: 139-143

9. Kannangara CC, Gough SP, Girnth C (1981) 6-Aminolevulinatesynthesis in greening barley. 2. Purification of enzymes. In GAkoyunoglou, ed, Proceedings of the Fifth International Pho-tosynthesis Congress, Vol 5. Balaban, Philadelphia, pp 117-127

10. Kannangara CG, Schouboe A (1985) Biosynthesis of 6-aminolev-ulinate in greening barley leaves. VII. Glutamate 1-semialde-hyde accumulation in gabaculine treated leaves. Carlsberg ResCommun 50: 179-191

11. Mau Y-HL, Wang W-Y (1988) Biosynthesis of 6-aminolevulinicacid in Chlamydomonas reinhardtii. Study of the transamina-tion mechanism using specifically labeled glutamate. PlantPhysiol 86: 793-797

12. Mauzerall D, Granick S (1956) The occurrence and determina-tion of 6-aminolevulinic acid and porphobilinogen in urine. JBiol Chem 219: 435-445

13. Rando RR (1977) Mechanism of the irreversible inhibition of 'y-aminobutyric acid-a-ketoglutaric acid transaminase by theneurotoxin gabaculine. Biochemistry 16: 4604-4610

14. Rando RR, Bangerter FW (1976) The irreversible inhibition ofmouse brain -y-aminobutyric acid (GABA)-a-ketoglutaric acidtransaminase by gabaculine. J Am Chem Soc 98: 6762-6764

15. Sawicki E, Hauser TR, Stanley TW, Elbert W (1961) The 3-methyl-2-benzothiazolone hydrazone test. Sensitive new meth-ods for the detection, rapid estimation and determination ofaliphatic aldehydes. Anal Chem 33: 93-96

16. Schneegurt MA, Beale SI (1988) Characterization of the RNArequired for biosynthesis of 6-aminolevulinic acid from gluta-mate. Purification by anticodon based affinity chromotographyand determination that the UUC glutamate anticodon is ageneral requirement for function in ALA biosynthesis. PlantPhysiol 86: 497-504

17. Soper TS, Manning JM (1982) Inactivation of pyridoxal phos-phate enzymes by gabaculine. Correlation with enzymatic ex-change of ,B-protons. J Biol Chem 257: 13930-13936

18. Urata G, Granick S (1963) Biosynthesis of a-aminoketones andthe metabolism of aminoacetone. J Biol Chem 238: 811-820

AVISSAR AND BEALE858



PYRIDOXAL PHOSPHATE IN 6-AMINOLEVULINIC ACID BIOSYNTHESIS

19. Wang W-Y, Gough SP, Kannangara CG (1981) Biosynthesis of6-aminolevulinate in greening barley leaves. IV. Isolation ofthree soluble enzymes required for the conversion ofglutamateto 5-aminolevulinate. Carlsberg Res Commun 46: 243-257

20. Wang W-Y, Huang D-D, Stachon D, Gough SP, Kannngara CG(1984) Purification, characterization, and fractionation of the6-aminolevulinic acid synthesizing enzymes from light-grownChlamydomonas reinhardtii cells. Plant Physiol 74: 569-575

21. Weinstein JD, Beale SI (1985) Enzymatic conversion of gluta-mate to 6-aminolevulinate in soluble extracts of the unicellulargreen alga, Chlorella vulgaris. Arch Biochem Biophys 237:454-464

22. Weinstein JD, Mayer SM, Beale SI (1987) Formation of 6-aminolevulinic acid from glutamic acid in algal extracts. Sep-aration into an RNA and three required enzyme componentsby serial affinity chromatography. Plant Physiol 84: 244-250

859



biosynthesisof tetrapyrrole pigment precursors1and fractionated by differential (nh4)2so4...

Documents