One-Carbon Generation in Saccharomyces Cerevisiae M

JouisNL OF BACTzIUOLOGY, Feb. 1977, p. 926-933 Vol. 129, No. 2 Copyright 0 1977 American Society for Microbiology Printed in U.S.A. "Active" One-Carbon Generation in Saccharomyces cerevisiae M. OGUR,* T. N. LIU,' I. CHEUNG, I. PAULAVICIUS,2 W. WALES, D. MEHNERT, AND D. BLAISE Department ofMicrobiology, Southern Illinois University, Carbondale, Illinois 62901 Received for publication 17 August 1976 A new mutation introducing a one-carbon requirement (e.g., formate) for the glycine-supplemented growth of a serine-glycine auxotroph (serl) was correlated with a lack of glycine decarboxylase activity. The presence of oxalate decarboxylase activity or glyoxylate decarboxylase activity did not overcome the one-carbon requirement. Another mutation characterized by the absence of oxalate decarboxylase activity did not introduce a one-carbon requirement. The presence and physiological significance of glycine decarboxylase activity in Saccharomyces are thus inferred. The primacy of the phosphorylated pathway oxylate by a glyoxylate decarboxylase (6) might to serine, glycine, and "active" one-carbon units also serve as a possible route of active one-car- in yeast strains growing in glucose minimal bon generation. media rests, in part, on the catabolite repres- The current study reports the presence of sion by fermentable substrates of an alterna- glycine decarboxylase, glyoxylate dehydrogen- tive pathway to glycine arising from nonfer- ase, glyoxylate decarboxylase, and oxalate de- mentable substrates and proceeding via the carboxylase activities in yeast and presents evi- tricarboxylic acid cycle to glyoxylate and gly- dence that a new mutation introducing a one- cine (16). Mutants blocked in the phosphoryl- carbon requirement (e.g., formate) for the ated pathway to serine are thus auxotrophic in growth of serine-glycine auxotrophs on glycine media supplied with repressing carbon sources was correlated with the lack of glycine-decar- (e.g., glucose), but slowly prototrophic in media boxylase activity. supplied with derepressing carbon sources (e.g., acetate). MATERIALS AND METHODS The pathway of "active" one-carbon genera- Yeast strains. Bakers' yeast was obtained from tion in the case of serine-glycine yeast auxo- Anheuser-Busch (St. Louis, Mo.). Other strains trophs growing in glycine-supplemented mini- were from the Carbondale collection: MO-11-48A a mal medium, or in the case of wild-type yeasts is a prototrophic haploid; MO-171-11B a serl is a growing on acetate minimal medium, appeared segregant derived from the outcross of strain 1453- to be uncertain and to require clarification. The 2B a add gal3 his8 met2 thr4 pet6 serl; MO-171- 11B-EMS-41 was derived from MO-171-11B by ethyl inference of the existence of such a pathway methane sulfonate (EMS) treatment and required was implicit in an earlier demonstration in formate for growth on glycine minimal mediuim; yeast that some label from [2-14C]acetate (9) MO-171-2B, a serl, is another segregant of the MO- and label from [2-'4C]glycine (8) were incorpo- 171 cross; 37892, aglul, is a glutamate auxotroph. rated into the 13-carbon of serine under appro- Media. (i) Standard complex medium. The stan- priate conditions. dard complex medium contained (grams per liter): D- Several possible pathways seemed reasona- glucose, 10; peptone, 3.5; KH2PO,, 2; MgSO, 7H,O, ble from experience with other materials: (i) 1; (NH4)2SO4, 2; dried yeast extract, 5. the decarboxylation of glycine to yield 5,10- (ii) Standard minimal medium. The standard minimal medium contained (per liter): D-glucose, 10 methylene FH4, NH3, and C02 by a glycine g, KH2PO4, 1 g; MgSO4* 7H2O, 0.5 g; (NH4)2,04, 1 g; decarboxylase system had been reported in bac- CaCl2, 0.3 mg; KI, 10 mg; choline chloride, 4 mg; teria (1, 2, 11, 15); (ii) the decarboxylation of inositol, 1 mg; nicotinic acid, 0.4 mg; calcium panto- oxalate to yield formate from glyoxylate by the thenate, 0.4 mg; pyridoxine * HCI, 0.4 mg; thia- combined action of glyoxylate dehydrogenase mine HCl, 0.4 mg; p-aminobenzoic acid, 0.4 mg; and oxalate decarboxylase had been reported biotin, 0.002 mg; FeSO4 *7H2O, 0.3 mg; (13, 14) in Pseudomonas oxalaticus strains in MnSO4-4H20, 0.04 mg; (NH4),,Mo70O24.4H20, 0.018 which glycine decarboxylase activity was not mg; Na2B407 10H20, 0.088 mg; CuSO4 5H2O, 0.04 mg; ZnSO4. 7H20, 0.31 mg; adjusted to pH 5.8 with observed; (iii) the direct decarboxylation ofgly- KOH. I Present address: P.O. Box 7902, University Station, Any changes in these media are specified in indi- Austin, TX 78712. vidual experiments. For the preparation of solid 2 Betz Laboratories, Somerton Road, Trevose, PA 19047. media, 25 g ofagar was added to 1 liter ofmedium. 926 VOL. 129, 1977 "ACTIVE" ONE-CARBON GENERATION 927 Growth of cultures. Cultures were incubated at mixed with 600 ml of well-washed glass beads (180- 300C on reciprocating shakers. Growth was esti- ,um diameter, Microbeads; Cataphote Division, mated colorimetrically in cultures inoculated into Ferro Corp., Cleveland, Ohio) in a Micro Mill (MV- either screw-capped matched Klett tubes or into 250- 6-3; Gifford Wood, Inc., Hudson, N.Y.), and yeast ml side arm flasks by measuring light absorbance in mitochondria were isolated by the procedure of Bal- a Klett-Summerson photoelectric colorimeter with a cavage and Mattoon (3). For breakage, the mill was blue 420-nm filter. operated at a gap setting of 0.040 inch (ca. 0.10 cm) Yeast enzyme,preparations with glyoxylate dehy- and a powerstat setting of 55 for 8 min, with pH drogenase, glyoxylate decarboxylase, and oxalate readjustment to 7.0 by 1 N NaOH. decarboxylase activities. Enzyme preparations from Assay of enzymatic activities. (i) Glyoxylate de- commercial bakers' yeast were begun with a 4-h hydrogenase activity. The glyoxylate dehydrogen- activation of the yeast cells by agitation in a 0.1% ase activity of yeast is not nicotinamide adenine glucose-2% lactate complex broth (10 g[wet weight] dinucleotide (NAD) dependent and was therefore of cells/100 ml of medium). Cells were harvested by assayed with a phenazine methosulfate-iodonitro- centrifugation and washed three times with distilled tetrazolium violet acceptor system based on the water and once with the cell breakage buffer (0.05 M method reported for succinic dehydrogenase (5). The sodium phosphate, pH 7.6, for glyoxylate dehydro- reaction mixture contained (in a total volume of 3 genase and glyoxylate decarboxylase and pH 6.8 for ml): sodium phosphate (pH 7.6), 150 ,umol; phena- oxalate decarboxylase). A 50% cell suspension was zine methosulfate, 1.33 ,umol; iodonitrotetrazolium, subjected to ultrasonic treatment (model S110; 1.2 ,umol; sodium glyoxylate, 10 ,umol; and enzyme Bransop Sonic Power Co., Danbury, Conn.) for 4 preparation, 0.1 ml. The absorbance change at 540 min at a power -setting of 7, tuned for maximum nm was followed continuously in a Gilford recording amperage. Cell breakage (60 to 80% efficient) and all spectrophotometer (model 2000; Gilford Instrument procedures before the enzyme assay were performed Laboratories, Inc., Oberlin, Ohio). in the cold (0 to 4°C). The sonically treated material (ii) Assay of decarboxylase activities. The activi- was centrifuged (model RC2B; Ivan Sorvall, Inc., ties of glyoxylate, oxalate, and glycine decarboxyl- Norwalk, Conn.) for 30 min at 48,000 x g. The pellet ase were assayed by using the appropriate 14C sub- was discarded and the supernatant fluid containing strates labeled either uniformly or specifically in the glyoxylate dehydrogenase and glyoxylate decarbox- first or second carbon (in the case of glyoxylate and ylase activities was dialyzed against three changes glycine). of the assay buffer. The dialyzed enzyme was frac- In preliminary assays with [14C]oxalate, the loss tionated with increasing ammonium sulfate concen- of 14C as a volatile product was estimated by differ- tration, and the fraction that sedimented at 48,000 x ence. This was checked by chromatographic separa- g between 50 and 80% saturation was collected. The tion of the incubation mixture and estimation of the 50 to 80% pellet was suspended and dialyzed for 2 h number of counts on a radiochromatogram in the against 2 liters ofthe cell breakage buffer and either new product peak that had the same Rf value as used immediately or stored at -20°C. The stored formate in six solvent systems. Stoichiometric enzyme preparation lost very little glyoxylate dehy- equivalence between volatile counts lost and for- drogenase or glyoxylate decarboxylase activity after mate counts formed was demonstrated by using a 1 month at -20°C. radiochromatogram strip scanner equipped with a Oxalate decarboxylase activity was found to be digital integrator (Actigraph III; Nuclear-Chicago unstable under these conditions and was generally Corp., Des Plaines, Ill.). assayed immediately in the 48,000 x g supernatant The count-loss assays were confirmed by a 14CO2 of the broken cell preparation. trapping assay applied to all three decarboxylase Enzyme preparations from laboratory yeast activities in which counts were estimated with a strains were begun by fresh transfer from a refriger- liquid scintillation spectrometer (model Mark III; ated storage slant to a working slant, which, after 24 Nuclear-Chicago Corp., Des Plaines, Ill.). The incu- h, was used to inoculate liquid medium in the appro- bation mixtures were pipetted into plastic analyzer priate volume to yield the desired cell harvest from cups (Lancer-Sherwood Medical Industries, Inc., St. 24- to 48-h cultures. Harvested cells were treated as Louis, Mo.). The cups were sealed with caps fitted above to produce the cell-free enzyme preparations. with 10-mm fiber glass prefilter disks (Millipore Yeast mitochondrial preparations with glycine Corp., Bedford, Mass.) impregnated with 0.05 ml of decarboxylase activity. Yeast cells were activated a 1 M solution of hydroxide of hyamine (Packard by incubation for 12 h in 10 liters of complex liquid Instrument Co., Downers Grove, Ill.) to trap evolved medium containing 0.1% glucose and 3% ethanol in CO2. Reactions were initiated by the addition of a fermentor with vigorous aeration (The VirTis Co., either enzyme preparation, mitochondria, or cell Inc., Gardiner, N.Y.). The culture was concentrated suspension. After the desired incubation period at with a Westfalia separator (Centrico, Inc., Engle- 30°C, the reaction was stopped by the addition of 0.1 wood, N.J.), harvested, and washed by centrifuga- ml of 1 N HCI to complete the transfer of "4CO2 from tion (International refrigerated centrifuge model the incubation mixture to the trapping disk. The PR-2; International Equipment Co., Needham, disk was then transferred to a scintillation vial con- Mass.). A total of 400 g of cells, suspended in the taining 10 ml of Bray solution (4) and counted under hypertonic breakage buffer [0.6 M mannitol-10-4 M appropriate conditions. The counting efficiency was ethylenediaminetetraacetate-0.2% serum albumin- estimated by internal standardization to be 76% 0.03 M tris(hydroxymethyl)aminomethane-maleate with a [14C]toluene standard (Packard Instrument buffer (pH 7.0)] to a volume of 600 ml, was Co.). The CO2 trapping efficiency was estimated to 928 OGUR ET AL. J. BACTERIOL. be 98% by acidification of sodium [14C]bicarbonate. mal medium was observed compared with the The incubation mixture for the oxalate decarbox- absence of an increase with incubation in glu- ylase assay contained: buffer-0.05 M phosphate cose minimal liquid medium. (pH 6.8), 50 gl; substrate- [U-14C]oxalate (5 jLCi/3 that ml), 10 ,ul (specific activity, 77 mCi/mmol); cofactor The slow growth on acetate suggested it mixture-ddithiothreitol, 0.2 jumol; coenzyme A, 0.1 was serving as the obligatory, but rate-limit- ,umol; adenosine 5'-triphosphate, 2 Lmol; MgSO4 ing, source of both one- and two-carbon inter- 7H20, 0.01 Mmol; thymine pyrophosphate, 0.1 ,mol mediates for glycine, serine, and active one- 20 ,ul); enzyme-43.5 mg of total protein per ml (40 carbon biosynthesis. A number of possible pre- ,.l); total, 120 ,ul. cursors for these syntheses (formate, glycine, The incubation mixture for the assay of glycine glyoxylate, and oxalate) were tested by esti- decarboxylase activity in intact yeast cells con- mating their ability to enhance the slow growth tained: substrate- [14C]glycine, 50 ,uCi/3 ml (100 p1) of a serl strain on the acetate minimal me- (specific activity, 50 mCi/mmol); enzyme prepara- dium. The results are shown in Fig. 1. All tion-intact yeast cells, 109 cells/ml (100 p1l). Both substrate and cells were prepared in 0.1 M phos- showed some degree of stimulation, including phate buffer (pH 6.0). oxalate, which, as the sole C source, was inef- The incubation mixture for the assay of glycine fective. decarboxylase activity in intact yeast mitochondrial To test for possible routes by which these preparations contained: substrate- [14C]glycine, 50 stimulatory effects might be explained, a series 4CiI3 ml (50 mCi/mmol) (100 ,l); cofactor solution- of enzymatic activities was asayed. PALP, 10 ,umol/ml, and FH4, 0.2 ,mol/ml (50 p.l); Glyoxylate dehydrogenase and oxalate de- yeast mitochondrial suspension-30 mg of protein carboxylase activities in yeast enzyme prepa- per ml (100 Ml); total, 250 Al. The substrate and rations. The conversion ofglyoxylate to oxalate cofactor solution were prepared in 0.01 M phosphate (pH 7.0) containing 0.65 M mannitol. was demonstrated both by the colorimetric as- The incubation mixture for the assay of glyoxyl- say for glyoxylate dehydrogenase activity and ate decarboxylase (no attempt was made to distin- by following the disappearance, chromato- guish between glyoxylate decarboxylase and glyoxylate carboligase activities) activity contained: buffer-0.05 M sodium phosphate (pH 7.6) (40 ,l); substrate- [14C]glyoxylate, 50 ,Ci/3 ml (20 Iul) (specific activity, 7.2 mCi/mmol); cofactors-thiamine pyrophosphate, 50 Amol/ml (20 Il), and MgSO4. 7H20, 7 ,umol/mol (20 ,l); yeast enzyme preparation-dialyzed 50 to 80% ammonium sulfate fraction, 41.5 mg ofprotein per ml (20 ,ul); total, 120 IAl. RESULTS Growth of the serl mutant. Growth charac- teristics ofyeast strains bearing the serl mutation involved the total requirement for serine or glycine on media containing catabolite-repressing C sources (e.g., glucose) and slow prototrophic growth on derepressing C sources (e.g., acetate), a behavior described as conditional auxotrophy (16). This was reconfirmed by direct plating of a serl strain (MO-171-11B) on acetate minimal agar and glucose minimal agar. Whereas no colonies formed on glucose minimal agar (unless supplemented by serine or glycine), small I .1 colonies at frequencies comparable to 100% via- 0 10 20 30 40 s0 bility did form on acetate minimal agar, reach- TIME (HRS) ing an average size ofapproximately 0.5 mm in FIG. 1. Enhancement of the slow growth on ace- diameter in 4 days of plate incubation at 30°C. tate minimal medium ofstrain MO-171-11B (a serl) Slow of in by various one- or two-carbon additives. Symbols: *, growth the serl mutant acetate mini- acetate minimal; 0, acetate minimalplus glycine; *, mal medium was also confirmed by incubating acetate minimal plus formate; 0, acetate minimal its cells in acetate minimal liquid medium, plus oxalate; A, acetate minimal plusglyoxylate. The sampling at 0, 12, 24, and 48 h, and plating onto concentration of the additive was 100 mg/liter. control complex agar. An increase in the num- Growth on 600 mg ofthe additive per liter as sole C ber ofcolonies after incubation in acetate mini- source was negligible. VOL. 129, 1977 "ACTIVE" ONE-CARBON GENERATION 929 graphically, of the substrate peak (glyoxylate) TABLE 1. R, valuesa of substrate and product of and the reciprocal appearance of a new product glyoxylate dehydrogenase activity peak (oxalate). Using [14C]glyoxylate, the prod- R, value uct of the incubation with the yeast enzyme preparation was found to have the same Rf Solvent system Glyoxyl- Oxalate Meta- value as [14C]oxalate in 10 different solvent sys- ate stan- bolic tems (Table 1). dard standard product The conversion to n-Butanol-acetone- 0.48 0.12 0.12 metabolic of oxalate for- water-diethylamine mate and CO2 was demonstrated by incubating (20:20:10:3)b ['4Cloxalate with a yeast enzyme preparation to Ethanol-diethylamine 0.87 0.76 0.74 which appropriate cofactors similar to those (80:20) found effective in the oxalate decarboxylase as- Ethyl acetate-formic 0.51 0.34 0.33 acid-water (10:3:2) say inP. oxalaticus (13) had been added. Omis- Ethanol-70% ethylamine- 0.51 0.00 0.00 sion of the added cofactors from the yeast en- water (80:10:19) zyme system resulted in a loss of more than 90% Ethanol-70% ethylamine 0.43 0.04 0.04 of the activity demonstrated in the complete (90:10) Ethanol-n-butanol- 0.60 0.33 0.32 system. The formate product was identified in water (4:4:1) six solvent systems (Table 2). One-half of the Ethanol-26% ethylamine 0.58 0.18 0.177 counts lost from [14C]oxalate were recovered in (80:20) the [14C]formate peak, the remainder being re- Ethanol-13% ethylamine 0.66 0.11 0.11 (80:20) covered as 14CO2. Ethanol-ammonia- -C 0.22 0.22 Glyoxylate decarboxylase activity in yeast water enzyme preparations. The ability to decarbox- Water-saturated phenol -C 0.40 0.39 was ylate glyoxylate demonstrated first by in- a Chromatographic separation on paper strips (no. 589, cubating uniformly labeled ['4C]glyoxylate orange ribbon C; Schleicher and Schuell, Keene, N. H.). with the 50 to 80% ammonium sulfate fraction b The numbers in parentheses represent the ratio of the of a broken yeast cell preparation. The 14CO2 volume of the solvents. produced dropped markedly with the omission SStreak. of thymine pyrophosphate or enzyme and to a TABLz 2. substrate and lesser extent when Mg2+ was omitted. The addi- Rf valuesa of product of tion of NAD did not enhance the reaction, and oxalate decarboxylase activity the addition of FH4 appeared to inhibit the R, value activity partially at this level of purification (Table 3). Solvent system Oxalate Formate Un- standard standard Supporting evidence for the existence of a productknown glyoxylate decarboxylase activity distinct from n-Butanol-acetone- 0.12 0.55 0.54 any possible decarboxylation via the tricar- water-diethylamine boxylic acid cycle was obtained by employing (20:20:10:3)b C2-labeled and a tricarbox- Ethanol-ammonia- 0.22 0.50 0.50 Cl- and glyoxylate water (85:5:15) ylic acid cycle mutant blocked at aconitase. Re- Ethanol-ethylamine 0.04 0.47 0.48 sults shown in Table 4 indicate that the amount (90:10) of CO2 evolved and trapped with [2-'4C]glyoxyl- Ethanol-butanol-water 0.33 0.70 0.69 ate as substrate was reduced to the 20% level as (5:4:1) Ethanol-13% ethylamine 0.11 0.71 0.70 compared with [1-'4C]glyoxylate when the two (80:20) substrates were introduced at equal concentra- Ethanol-butanol-water- 0.20 0.50 0.50 tion and specific activities. No attempt was diethylamine made to distinguish between glyoxylate decar- (40:50:10:10) boxylase and glyoxylate carboligase activities. a Separation was on paper (see footnote a of Table 1). Glycine decarboxylase activity of intact bThe numbers represent the ratio of the volume of the yeast cells and mitochondrial preparations. solvents. Our early attempts to demonstrate a glycine cleavage reaction in broken yeast cell prepara- labeled glycine, as suggested (10) for liver ho- tions by the method of Sagers and Gunsalus mogenates. Results shown in Table 5 indicate (15) were not successful. that the 14CO2 produced from [2-'4C]glycine was Assuming possible inactivation of the en- reduced to the 7% level as compared with the zyme in the cell breakage isolation procedure, 4CO2 produced from [1-_4C]glycine. an attempt was made to demonstrate glycine Attempts to demonstrate the same activity in decarboxylase activity in intact yeast cells as yeast enzyme preparations derived from a num- the "4CO2 differentially produced by Cl- and C2- ber of different cell breakage procedures re- 930 OGUR ET AL. J. BACTCRIOL.

TABLE 3. Glyoxylate decarboxylase activity of mentation was mutagenized by EMS treat- enzyme preparations from bakers' yeasta ment, and clones growing on serine, but not on Reaction mixture "4CO2 trapped (cpm) glycine unless supplemented by formate, were selected. One of the mutant isolates (MO-171- Complete system 45,088 11B-EMS-41) studied more extensively ex- _TPPb 3,598 -Mg2+ 28,188 hibited growth in liquid media, which con- -Enzyme 1,224 firmed the selection procedure on solid media. +NAD 44,898 Figure 2 compares the growth of the serl and +FH4 29,999 the serl-EMS-41 strains on glucose media, the new for a demonstrating requirement glycine Incubation conditions were 90 min at 30°C. The and formate and suggesting that a new muta- concentration of NAD and FH4 were 10 and 0.1 /Amol/ml, respectively. tion had been introduced in a physiologically b TPP,Thymine pyrophosphate. significant pathway of active one-carbon biosynthesis. TABLE 4. Glyoxylate decarboxylase activity in a Decarboxylase activity of the EMS-41 mu- tricarboxylic acid cycle mutant (glul)a tant. To determine whether the requirement for one-carbon supplementation (formate) re- Reaction mixture (4COS trapped lated to any loss ofability to decarboxylate two- carbon substrates, assays were carried out (Ta- [1-'4C]glyoxylate-complete system 70,997 [2-'4C]glyoxylate-complete system 12,261 ble 8). Two decarboxylase activities (glycine [1-14C]glyoxylate-complete system minus 520 and oxalate) present in the serl mutant were enzyme lacking in the EMS-41 mutant. The glyoxylate * glu-1 strain, no. 37892, a glu-1. The incubation time decarboxylase activity was comparable in both was 90 min. (as was the glyoxylate dehydrogenase activity). Because the assay systems for the two miss- TABLz 5. Glycine decarboxylase activity assayed ing decarboxylase activities required different with intact yeast cells" cofactors, the probability existed that two different enzyme systems might be involved. Res- Labeled glycine '4CO2 trapped (cpm) [1-14C]glycine 7,305 TABLE 6. Glycine decarboxylase activity of isolated [2-'4C]glycine 456 yeast mitoChondriaa Minus cells 311 Reaction mixtureb 14CO, trapped a Strain MO-171-11B. The incubation time was (cpm) for 60 min at 30°C. The cell suspension was 10" cells [1-_4C]glycine-complete system 12,791 per incubation vessel. [1_-4C]glycine minus cofactors 7,098 [1-_4C]glycine minus enzyme 2,104 sulted in failure until recourse was had to a [2-'4C]glycine-complete system 1,685 yeast mito- procedure yielding tightly coupled a The incubation time was 3.5 h at 30°C; protein, chondria (3). The glycine decarboxylase activ- 30.3 mg/ml. ity of mitochondria prepared from activated b [1-_4C]glycine (specific activity, 50 mCi/mmol); bakers' yeast is summarized in Table 6. A ma- [2-'4C]glycine (specific activity, 50 mCi/mmol). jor difference in 14CO2 production between Cl- and C2-labeled glycine as substrates was ob- TABLz 7. Glycine decarboxylase activity ofdialyzed served and only a low level of nonenzymatic enzyme preparation from broken mitochondria of decarboxylation with Cl glycine. Omission of bakers' yeasta the added cofactors (PALP and FH4) resulted in Tratment "4CO, trapped (cpm/h) a 47% decrease in activity. When yeast mito- Complete 29,139 chondria isolated in the tightly coupled state -NAD 18,861 were broken by gentle grinding in a glass ho- -FH4 6,222 mogenizer and dialyzed against buffer, a prepa- -PALP 2,715 ration with increased cofactor dependence was -Cofactors 2,114 obtained (Table 7). Elimination of NAD, FH4, -Enzyme 512 and PALP produced a decrease in activity greater than 90%. a The specific activity of the [14C]glycine was adjusted to 0.1 mCi/mmol by the addition of cold car- Induction and isolation of a new mutation rier glycine. The incubation mixture contained: lesion producing a requirement for added for- NAD, 0.4 ,umol; FH4, 0.4 ,umol; PALP, 0.15 ,umol; [1- mate in serl mutants growing on glycine. A "4C]glycine, 0.5 ,uCi; dithiothreitol, 6 Amol; sodium serl mutant strain (MO-171-11B) capable of phosphate, pH 7.3, 30 ,umol; protein, about 12 mg; growing with either serine or glycine supple- total volume, 0.6 ml. VOL. 129, 1977 "ACTIVE" ONE-CARBON GENERATION 931 mate (data not shown). Two segregants in each tetrad lacked oxalate decarboxylase activity and two lacked glycine decarboxylase activity, but these lesions were separated by genetic segregation, suggesting independent two-gene control. Two segregants from each of eight tetrads required formate supplementation for growth on glycine and lacked glycine decarboxylase activity in the intact cell assay. To rein- (A force these results, one complete tetrad was subjected to the mitochondrial glycine decarboxylase activity assay (Table 10). Again, the I-- without formate I.- inability to grow on glycine .i-J supplementation correlated with the lack of glycine decarboxylase activity. DISCUSSION The present study considered three possible enzymatic systems never before reported in yeast (but reported in other materials) as rea- sonable possibilities for a role in active one- carbon generation from two-carbon substrates in yeast. These involved decarboxylase reac- 0 4 8 12 16 20 24 28 32 36 tions for glycine, glyoxylate, or oxalate. We had TIME (HOURS) previously demonstrated that glycine and gly- FIG. 2. Growth of the serl and serl-EMS-41 mu- oxylate enhanced the growth rate of the serl tants on supplemented minimal media. Symbols: 0, auxotroph on acetate media. In the current serl on minimal plus glycine medium; *, serl-EMS- work, oxalate was also found to enhance 41 on minimal plus glycine medium; 0, serl on growth of both the serl mutant and bakers' minimal plus glycine plus formate medium; *, serl - yeast strains. This stimulated a search for and EMS-41 on minimal plus glycine plus formate me- led to the discovery of new enzyme activities of dium. oxalate formation and decomposition in yeast (glyoxylate dehydrogenase and oxalate decar- TABLE 8. Decarboxylase activity in serl and serl- boxylase). The glyoxylate dehydrogenase activ- EMS-41 mutants ity, which is neither NAD nor NADP depend- Oxalate de Activity of Glycine de- ent in yeast (like the yeast lactic dehydrogen- Enzyme pre- carboxylase loyae carboxylase ase), was first detected by the conversion of pared from: (cpm/mg per de(carbxycpmlOpeh ['4C]glyoxylate to [14C]oxalate. The labeled as- h) ae(cpm/mg cells per h) per h) say was supplemented by a spectrophotometric serl-EMS-41 0 50,216 111 assay employing phenazine methosulfate and serl 487 45,043 2,589 p-iodonitrotetrazolium violet. Our unpublished No enzyme 0 11 79 work on the partial purification and characterization of the yeast glyoxylate dehydrogenase activity has indicated that it is distinct from olution of the two activities was attempted by either the D- or ilactate dehydrogenase activi- genetic segregation in outcross. ties. Oxalate decarboxylase activity was also Segregation of the lesions in decarboxylase discovered, using [14C]oxalate incubated with a activities. The derived formate-requiring clone yeast enzyme preparation and appropriate co- (MO-171-11B-EMS-41) was crossed to a for- factors similar, but not identical, to the P. oxal- mate-independent strain (MO-171-2B) and a aticus system. Evolved 14CO2 was trapped in family homozygous for serl, but heterozygous alkali and counted, and [14C]formate was iden- for the new formate requirement, was con- tified after chromatographic separation of the structed. Segregants were analyzed for the incubation mixtures. Glycine and glyoxylate three decarboxylase activities as well as the decarboxylase activities were also found in serine, glycine, and formate requirements. The wild-type and serl mutant yeasts. results for eight tetrads of this cross are shown The proof of the physiological significance of in Table 9. All segregants failed to grow on one or more of the decarboxylase activities was minimal medium and grew on both minimal sought in the induction of and selection for a plus serine or minimal plus glycine plus for- mutant blocked in both the primary and sec- 932 OGUR ET AL. J. BACTERIOL. TABLE 9. Genetic segregation ofthe formate requirement and the decarboxylase lesions in the outcross ofthe EMS-41 mutant Glycine decarboxyl- Growth on minimal Oxalate Strain Mating type ase activity + glycine (no for- activitydecarboxyl- Parents MO-171-2b, a + Mo-171-llB-EMS-41 a Segregants MO-301-la a lb a + + + lc a ± ± ld a + 301-2a a + + 2b a + + 2c a + 2d a + 301-3a a + + + 3b a 3c a + 3d a + + 301-4a a + 4b a + + 4c a + 4d a + + 301-5a a + + + 5b a 5c a + + + 5d a 301-6a a 6b a + 6c a + + + 6d a + + 301-7a a + 7b a 7c a + ± + 7d a + + 301-8a a + + 8b a + + 8c a ± 8d a +

ondary pathways of active one-carbon genera- of each other, and probably reside in different tion. This was achieved by mutagenizing a serl enzyme systems under separate genetic control. strain (blocked in the phosphorylated pathway The new requirement for formate in serl at glutamate:P-OH-pyruvate transaminase) strains growing on glycine segregated with the with EMS and selecting for a clone that would lack of glycine decarboxylase activity and inde- fail to grow on glucose minimal plus glycine pendently of the lack of oxalate decarboxylase medium unless supplemented by an exogenous activity. It was thus possible to infer that the one-carbon source (formate). One such clone glycine decarboxylase activity is on the alterna- (MO-171-11B-EMS-41) was assayed and found tive route of active one-carbon generation in to possess glyoxylate dehydrogenase and decar- yeast, a route that involves the isocitrate lyase, boxylase activities, but to lack glycine and oxa- the alanine:glyoxylate transaminase, and the late decarboxylase activities. It would have glycine decarboxylase systems. been tempting to assume one enzyme system In summary, prototrophic yeasts growing on governing the decarboxylation of both glycine glucose media appear to generate serine, gly- and oxalate had the cofactor requirements not cine, and active one-carbon fragments princi- been different. pally via the phosphorylated pathway arising Genetic methods indicated that glycine and from Embden-Meyerhof pathway intermedi- oxalate decarboxylase activities segregated in ates because the alternative pathway is catabo- regular Mendelian fashion, but independently lite repressed almost completely (by a high glu- VOL. 129, 1977 "ACTIVE" ONE-CARBON GENERATION 933 TABLE 10. Genetic segregation ofglycine Metabolism and Digestive Diseases and grant 09-04-08 of decarboxylase activity ofmitochondria isolated from the Office of Research and Projects, Graduate School, the segregants of a tetrad Southern Illinois University. Glycine decarboxylasea Growth on activity -14CO2 trapped minimal + (cpm/3 h) Segregant glycine (no LITERATURE CITED formate) [1-_4C] [2-14C] glycine glycine 1. Baginsky, M. L., and F. M. Huenneken. 1966. Electron MO-301-la - 3,i54 103 transport function of a heat-stable protein and flavo- MO-301-lb + 19,935 228 protein in the oxidative decarboxylation of glycine by MO-301-lc + 12,551 273 Peptococcus glycinophilus. Biochem. Biophys. Res. MO-301-ld - 1,024 258 Commun. 23:600-611. 2. Baginsky, M. L., and F. M. Huenneken. 1967. Further -Enzyme 1,039 212 studies on the electron transport proteins involved in a [1-14C]glycine (specific activity, 50 mCi/mmol); oxidative decarboxylation of glycine. Arch. Biochem. Biophys. 120:703-709. [2-14C]glycine (specific activity, 50 mCi/mmol). 3. Balcavage, W. X., and J. R. Mattoon. 1968. Properties of Saccharomyces cerevisiae mitochondria prepared cose at isocitrate and ala- by mechanical method. Biochim. Biophys. Acta concentration) lyase 153:521-530. nine:glyoxylate transaminase. In acetate me- 4. Bray, G. A. 1960. A simple efficient liquid scintillator dia, on the other hand, the enzymes of the for counting aqueous solutions in a liquid scintilla- alternative pathway are derepressed, and car- tion counter. Anal. Biochem. 1:279-285. to flow via the acid 5. Criddle, R. S., and G. Schatz. 1969. Promitochondria of bon appears tricarboxylic anaerobically grown yeast. I. Isolation and biochemi- cycle and the three enzyme systems enumer- cal properties. Biochemistry 8:322-334. ated to glycine, active one-carbon generation, 6. Davies, D. D., and R. J. Corbett. 1969. Glyoxylate de- and serine. carboxylase activity in higher plants. Phytochemis- Thus, the glyoxylate system may be viewed try 8:529-542. 7. DeBoiso, J. F., and A. 0. M. Stoppani. 1963. The bio- as playing an anaplerotic role not only in gener- synthesis of serine in baker's yeast. Biochim. Bio- ating four-carbon substrates for continuing tri- phys. Acta 78:551-553. carboxylic acid cycle function, but also in ac- 8. DeBoiso, J. F., and A. 0. M. Stoppani. 1967. Metabo- tive one-carbon generation for protein and nu- lism of serine and glycine in baker's yeast. Biochim. Biophys. Acta 148:48-59. cleic acid biosynthesis during growth on tri- 9. Gilvarg, C., and K. Bloch. 1951. The utilization of carboxylic acid cycle substrates. The current acetic acid for amino acid synthesis in yeast. J. Biol. work provides experimental confirmation for a Chem. 193:339-346. This 10. Kaiya, T. L. M., R. E. Corbell, and E. Eggermont. possibility suggested by Kornberg (12). 1974. A block in glycine cleavage reaction as a com- report has concerned itself principally with ex- mon mechanism in ketotic and non-ketotic,hypergly- periments relevant to the demonstration of the cinemia. Pediatr. Res. 8:721-723. alternative pathway in yeast and has not dealt 11. Klein, S. M., and R. D. Sagers. 1967. Glycine metabo- with the lism. III. A flavin-linked dehydrogenase associated the mechanism by which yeast glycine with the glycine cleavage system in Peptococcus glyci- decarboxylase system transfers the alpha car- nophilus. J. Biol. Chem. 242:297-300. bon of glycine to FH4. Continuing work on the 12. Kornberg, H. L. 1966. The role and control of the glyox- purification and characterization of the four ylate cycle inEscherichia coli. Biochem. J. 99:1-11. enzyme systems and their 13. Quayle, J. R., E. B. Keech, and G. A. Talor. 1961. physiological signifi- Carbon assimilation by Pseudomonas oxalaticus cance will be published separately. The possi- (OXI). 4. Metabolism of oxalate in cell-free extracts bility that mutants with lesions in glycine de- of the organism grown on oxalate. Biochem. J. carboxylase and oxalate decarboxylase activi- 78:225-236. ties may serve as models for the study of gly- 14. Quayle, J. R., and G. A. Talor. 1961. Purification and properties of glyoxylate dehydrogenase. Biochem. J. cine and oxalate accumulation in animals (e.g., 78:611-618. nonketotic hyperglycinemia, hyperoxaluria, 15. Sagers, R. D., and I. C. Gunsalus. 1961. Intermediary kidney stones, etc.) has not escaped our consid- metabolism of Diplococcus glycinophilus I. Glycine eration. cleavage and one-carbon interconversions. J. Bacte- riol. 81:541-549. ACKNOWLEDGMENTS 16. Ulane, R., and M. Ogur. 1972. Genetic and physiologi- This study was supported by Public Health Service grant cal control of serine and glycine biosynthesis in Sac- 5 R01 AM 14768-02 from the National Institute of Arthritis, charomyces. J. Bacteriol. 109:34-43.