Bacterial Metabolism of Mevalonic Acid'MAJID A. SIDDIQI' AND VICTOR W. RODWELL3
Department of Biochemistry, School ofMedicine, University of Californzia, San Francisco Medical Center,San Francisco, California
Received for publication 19 August 1966
ABSTRACT
Soluble cell-free extracts of actinomycete S4 grown on media containing mev-alonate catalyze acetoacetate formation from mevalonate, mevaldate, and f-hy-droxy-3-methylglutaryl-coenzyme A (CoA). Conversion of mevalonate to aceto-acetate involves formation of free f3-hydroxy-f3-methylglutaryl-CoA, but not freemevaldate. The reaction favors mevalonate oxidation, and nicotinamide adeninedinucleotide, rather than nicotinamide adenine dinucleotide phosphate, acts asoxidant.
The biosynthesis of mevalonate in yeast andliver has been studied in detail in several labora-tories, notably those of H. Rudney, J. W. Porter,and F. Lynen. In liver, two reaction sequenceslead to mevalonate from acetyl-coenzyme A(CoA). One, catalyzed in part by microsomalenzymes (18), involves free acetoacetyl-CoA andreductive deacylation of free 3-hydroxy-3-methyl-glutaryl-CoA. Analogous reactions lead to thesynthesis of ,3-hydroxy-f-methylglutaryl-CoA(23, 24) and mevalonate in yeast, although quiterecently acyl carrier protein has been implicatedas a possible cofactor (19). The yeast oxidore-ductase catalyzing reduction of f3-hydroxy-f-methylglutaryl-CoA to mevalonate has been ex-tensively purified, and its mechanism has beenstudied (7).The second reaction sequence was initially
demonstrated in pigeon liver (3, 4), but occursalso in rat liver (G. M. Fimognari and V. W.Rodwell, Abstr. Intern. Congr. Biochem., 6th,New York, 1964, p. 573). Malonyl-CoA appearsto be the only free intermediate, and the enzymesinvolved are closely associated with those of fattyacid synthesis (4).Although mevalonic acid was originally dis-
covered as a growth factor for lactobacilli (25,28), little is known of its metabolism in bacteria.This paper describes a soil microorganism capableof growth with mevalonate as the sole source of
l Data from a thesis submitted by Majid A. Siddiqiin fulfillment of the requirements for the Ph.D. de-gree, University of California, San Francisco.
2 Present address: Muslim University, Aligarh,India.
3 Present address: Department of Biochemistry,Purdue University, Lafayette, Ind.
carbon. The kinetic properties of mevalonate:nicotinamide adenine dinucleotide (NAD) oxi-doreductase (acylating CoA) obtained from thisorganism (actinomycete S4) and from Pseudo-monas Ml have been described elsewhere (9).[Actinomycete S4 was referred to as Mycobacte-rium S4 in our previous publications (9, 21).]
MATERIALS AND METHODSChemical. Reagents obtained commercially in-
cluded the following: NAD, nicotinamide adeninedinucleotide phosphate (NADP), reduced nicotina-mide adenine dinucleotide (NADH2), and tris(hy-droxymethyl)aminomethane (Tris) (Sigma ChemicalCo., St. Louis, Mo.); CoA (Pabst Laboratories, Mil-waukee, Wis.); Celite 545 (Johns Manville Co.,Chicago, Ill.); ethylenediaminetetraacetic acid(EDTA), f3-hydroxy-jl-methylglutaric acid, and thedibenzylethylenediammonium salt of DL-mevaldateacetal (Mann Research Laboratories, New York,N.Y.); DL-mevalonic acid lactone (Nutritional Bio-chemicals Corp., Cleveland, Ohio); methacrylic acidand j0,fl-dimethylacrylic acid (Calbiochem); and thedibenzylethylenediammonium salt of DL-mevalonate-2-C14 (Volk Radiochemical Co., Chicago, Ill.).
DL-Mevalonic acid lactone was prepared from thedibenzylethylenediammonium salt by passage througha short Dowex 50 (H+) column (15), and was con-verted to potassium mevalonate with a slight molarexcess of KOH. After incubation at 37 C for 30 minto insure saponification, the pH was adjusted to 7.0.DL-Mevaldic acid was prepared just before use fromthe dibenzylethylenediammonium salt of mevaldateacetal (15). 63-Hydroxy-/3-methylglutaric acid anhy-dride (mp = 102-103 C, uncorrected) was synthe-sized according to Hiltz et al. (12). fl-Hydroxy-,3-methylglutarate hydroxamate was prepared from theanhydride by the general method of Lipmann andTuttle (13). ,3-Hydroxy-,3-methylglutaryl-CoA was pre-pared from the anhydride and CoA (14). Its purity
was evaluated according to Durr and Rudney (7) andby comparison of the absorbance at 260 m, withhydroxamate color (H. Rudney, personal communica-tion). The molar ratio of adenine absorption tohydroxamate color was 0.73. Higher ratios were notanticipated, since the stated purity of the CoA usedwas 75%. Acetoacetic acid was prepared from ethyl-acetoacetate (20). Crystalline L-lactate:NAD oxido-reductase (1.1.1.27), with a specific activity of 72,umoles per min per mg, was prepared from beef heart(17).Paper chromatography was by descending develop-
ment in most cases. Purification of acetone 2,4-dinitrophenylhydrazone to constant specific activitywas carried out on paper dipped in 10% ,B-phenoxy-ethanol in acetone and air-dried just prior tochromatography in n-heptane saturated with ,3-phen-oxyethanol (16). Free and acyl thiols were detectedby use of nitroprusside as described by Stadtman (22).
Bacteriological. Ionic medium had the followingcomposition: total phosphate, 11.2 mM; NO8-, 12.5mM; S04<, 0.26 mM; C1-, 0.034 mM; NH4+, 12.5 mM;Mg+', 0.24 mM; Fe+3, 0.0072 mM; Mn+2, 0.012 mM; andNa+, 0.034 mm. The final pH was 7.0. Cell concentra-tions were estimated turbidimetrically by use of aKlett photometer with a no. 66 (red) filter. Klett unitswere converted to bacterial density (milligrams, dryweight, per liter) by use of a standard curve preparedfrom a salt-free suspension of cells of known dryweight.
Isolation of organisms capable of growth on DL-mevalonate as sole organic nutrient. Four pure cultureswere isolated from local soil by stationary electiveculture at 25 C with the use of ionic medium con-taining 0.05% potassium DL-mevalonate, pH 7.0.These were transferred bimonthly to agar slants ofionic medium containing 0.4% DL-mevalonate. Cul-ture S4, which grew most rapidly and was used for allstudies, forms filamentous colonies, and is gram-positive, non-acid fast, nonmotile, and nonproteolytic.The cells are elongated with a tendency to branching.It forms a surface pellicle in stationary liquid medium,but is well dispersed in shaken culture. On the basisof these characteristics, S4 was assigned to the orderActinomycetales (Bergey's Manual).
Cell-free extract. Cells grown on 0.4% DL-mevalo-nate were harvested by centrifugation, washed, centri-fuged, and suspended in cold ionic medium to give 10to 20 mg of cells per ml (dry weight equivalent).Subsequent operations were carried out at about 5 C.The suspension was disrupted for 30 min in a 9-kcRaytheon sonic oscillator. The viscous supernatantliquid obtained after centrifugation (10,000 X g; 10min) to remove debris and unbroken cells is calledcrude extract. This extract was centrifuged (85,000 Xg; 30 min), and the pellet, which contains NADH2oxidoreductase (Table 6), was discarded. An amountof 136 mg of (NH4)2SO4 per ml of 85,000 X g super-natant liquid was then added. The precipitate obtainedafter centrifugation (15,000 X g; 15 min) was dis-carded. Protein precipitating on addition of a further158 mg of (NH4)2SO4 per ml of 85,000 X g super-natant liquid was collected by centrifugation anddissolved in 0.1 M potassium phosphate buffer (pH
7.0) to give the 25 to 50% (NH4)2SO4 fraction (Table6).
Chemical determinations. Mevalonic acid was esti-mated according to Lynen and Grassl (15), and mev-aldic and pyruvic acids, by the general procedure ofFriedman and Haugen (10) as described by Lynen(14). For determination of acetoacetic acid accordingto Walker (27), samples were deproteinized withtrichloroacetic acid and were adjusted to pH 5.0 with1.0 M sodium acetate buffer prior to analysis. CoAwas determined by the method of Grunert and Phillips(11), and ,B-hydroxy-,B-methylglutaryl-CoA, by thealkaline hydroxylamine procedure of Durr and Rud-ney (7). Protein was estimated by the method ofWaddell (26).To determine isotope incorporation into acetoace-
tate, the pH of protein-free supernatant liquids wasfirst adjusted to 5.0 with 1 M sodium acetate buffer.Acetoacetate was then decarboxylated to acetone byincubation at 30 C for 90 min with aniline hydrochlo-ride (2). Excess 0.5% 2,4-dinitrophenylhydrazine in2 N HCl was added, and the mixture was stored over-night at 4 C. The crude dinitrophenylhydrazones werewashed successively with water, 5% HCl, 10%0Na2CO3, and again with water. At this point dinitro-phenylhydrazones from actual incubation mixturesmelted at 124 to 125 C and showed no depression ofmelting point on mixing with authentic acetone 2,4-dinitrophenylhydrazone (mp = 124 to 125 C). Iso-lated acetone dinitrophenylhydrazone was dissolvedin ethyl acetate, and its concentration was determinedfrom the absorbance at 358 mu. It was then subjectedto repeated chromatography in n-heptane saturatedwith ,B-phenoxyethanol, eluted, counted, and rechro-matographed to constant specific activity.When carrier mevaldate was added and subse-
quently isolated as the 2,4-dinitrophenylhydrazone,the deproteinized reaction mixtures were divided inequal portions. Half was worked up for acetonedinitrophenylhydrazone, and the remainder, to whichno carrier acetone was added, was worked up in ananalogous manner but omitting the wash with Na2CO3.The dinitrophenylhydrazone was extracted from ethylacetate into Na2CO3, the carbonate was acidified, andthe product was re-extracted into ethyl acetate. Itsconcentration was then determined at 375 m,u.
Radioactive samples were counted on stainless-steel planchets, by use of a Nuclear-Chicago gas-flowcounter.
Manometric experiments were conducted at 30 C,pH 7.0, in a volume of 2.0 ml in 17-ml Warburg vesselsshaken at 110 strokes per minute. The center wellcontained 0.2 ml of 10% KOH.
RESULTSAlternate sources of carbon for growth of whole
cells. Several compounds were tested as sole or-ganic nutrients for growth in the hope of sheddinglight on the pathway of mevalonate dissimilation.Rapid growth occurred when mevalonate, glu-cose, acetate, L-malate, citrate, a-ketoglutarate,or L-glutamate was used as the sole source of car-bon, whereas fl-hydroxy-f-methylglutarate, meth-
acrylate, /,f3-dimethacrylate, or propionate didnot support growth.The cost of mevalonic acid prompted us to in-
vestigate whether enzymes of mevalonate oxida-tion were constitutive. The ability of washed cellsgrown for 18 to 24 hr on 0.4% mevalonate, glu-cose, or acetate to oxidize 12.5 mM DL-mevalonatewas tested in a conventional manometric experi-ment. Qo, values for mevalonate were 109, 5,and 2 ,ulters of 02 taken up per mg of cells (dryweight equivalent) per hr for mevalonate-, glu-cose-, and acetate-grown cells, respectively. Thus,one or more enzymes of mevalonate metabolismare present after growth on mevalonate, but notafter growth on glucose or acetate alone. How-ever, a separate experiment showed that cellsgrown on glucose plus mevalonate or on acetateplus mevalonate also oxidized mevalonate.
Stoichiometry of mevalonate oxidation in wholecells. The observed molar ratio of net oxygen con-
sumption to mevalonate disappearance approxi-mated the theoretical value of 7.0 when about40% of the initial DL-mevalonate had disappearedfrom the medium (Table 1). This suggests:
C6H1204 + 702 6CO2 + 6H20.
Detection ofacetoacetate and ca-ketoglutarate inthe supernatant liquid from growing cultures. A1-liter amount of cell-free supernatant liquid ob-tained after 24 hr of growth on 0.4% DL-mevalon-ate was concentrated under vacuum at 40 C. Anal-ysis indicated the presence of about 4 ,umoles ofacetoacetate and 0.6 ,umole of a-keto acids per
TABLE 1. Stoichiometry of mevalonate oxidations
DL-Mevalonate
Total MolarPer oxygen ratio of 02/
Initial Final Disap- cent consumed mevalonatebpeared uti-
-Washed cells grown on 0.4% DL-mevalonatewere used in a conventional manometric experi-ment at a final cell concentration of 1.7 mg of cellsper ml (dry weight equivalent). Oxygen consump-tion was followed for 6 hr. The data are correctedfor the small oxygen uptake observed in the ab-sence of added substrate. The flask contents werethen removed quantitatively, centrifuged, andwashed once with ionic medium, and the cell-freesupernatant liquid and washings were combinedand analyzed for residual mevalonate.
bExpressed as micromoles of 02 consumed permicromole of mevalonate utilized.
liter of original growth medium. Paper chromatog-raphy of the 2,4-dinitrophenyl-hydrazones fromconcentrated medium in n-butanol-95 % ethylalcohol-0.5 N NH40H (70:10:20, v/v) (8)showed two hydrazone areas which correspondedboth in RF value and in color after treatment withalcoholic KOH to authentic samples of acetonedinitrophenylhydrazone and a-ketoglutarate-di-nitrophenylhydrazone. Acetone 2,4-dinitrophen-ylhydrazone presumably arose by decarboxyla-tion of the acetoacetate derivative during paperchromatography. Recrystallization of the crudehydrazones from methanol yielded a yellow ma-terial, decomposition point at 214 to 215 C (un-corrected). Authentic a-ketoglutarate 2 ,4-dinitro-phenylhydrazone decomposed at 214 C. Theisolated hydrazone had a spectrum in alkali iden-tical to that of authentic a-ketoglutarate 2,4-di-nitrophenylhydrazone.
Conversion ofmevalonate to acetoacetate in cell-free extracts. That soluble extracts of mevalonate-grown cells catalyze conversion of mevalonate toacetoacetate was shown both by chemical analy-sis (Table 2) and by isolation of a radioactivederivative of acetoacetate (Tables 3 and 4). WithDL-mevalonate-2-C'4 as much as 35% of the totalisotope was found in the acetone derived fromacetoacetate by chemical decarboxylation (Table3).
TABLE 2. Precursors of acetoacetatea
Total sNetheEn- aceto- sisthof
Substrate added Amt zymeb acetate acitofformed acetate
m,umoles miAmolesDL-Mevalonate 410 - 0
410 + 85 85,3-Hydroxy-,3-methyl- 3,780 - 5
glutarate 3,780 + 2 0DL-Mevaldate 4,400 - 8
4,400 + 330 322,B-Hydroxy-,3-methyl- 1,750 - 8
glutaryl-CoA 1,750 + 505 497
a The indicated substrates plus the followingwere incubated at 30 C in 0.4 ml: 80 jLmoles of Trischloride, pH 8.0; 0.6 ,umole of CoA; 1.85 ,umoles ofNAD; and 0.8 mg of dialyzed crude extract pro-tein. CoA was omitted when ,-hydroxy-j8-methyl-glutaryl-CoA was the substrate. After 60 min, theentire sample was deproteinized with 0.1 ml ofcold 25% trichloroacetic acid, and samples of theprotein-free supernatant liquids were analyzedfor acetoacetate.
b Symbols: + = complete incubation; -= con-trol with enzyme omitted.
- Calculated as difference in millimicromolesformed in incubations with and without enzyme.
a Incubation of the following was done at 30 Cin 1.0 ml: 100 ;umoles of Tris chloride, pH 8.0;2.0,umoles of CoA; 3.0,umoles of NAD; 0.75 ,Amoleof DL-mevalonate-2-C'4 (specific activity = 1.15 X106 counts per min per gmole); 2.0 mg of dialyzedcrude extract protein; and either 0 (trial I), 9.4(trial II), or 47 (trial III) ,umoles of carrier DL-mevaldate. After 3 hr, reaction mixtures weredeproteinized with 0.1 ml of cold 25% trichloro-acetic acid. Control incubations had trichloraceticacid added before enzyme. The precipitates wereremoved by centrifugation and washed once withwater; the supernatant liquids and washings werecombined. Half was used to prepare mevaldate2,4-dinitrophenylhydrazone. To the remainder,25 to 50 ,umoles of carrier acetone was added, thepH was adjusted to 5.0 with 1 M sodium acetatebuffer, and the acetoacetate present was de-carboxylated to acetone. Acetone 2,4-dinitro-phenylhydrazone was then isolated, purified toconstant specific activity by paper chromatog-raphy, eluted, and counted.bCounted as acetone 2,4-dinitrophenyl-
hydrazone.- Counted as mevaldate 2,4-dinitrophenyl-
hydrazone.d Expressed as counts per minute calculated for
the entire sample.e Expressed as per cent of added radioactivity
found in mevaldate or in acetone derived fromacetoacetate.
f Actual samples counted 5 to 10 counts/minabove background.
Since mevaldate, j8-hydroxy-,3-methylglutarate,and /3-hydroxy-f3-methylglutaryl-CoA were allpossible intermediates between mevalonate andacetoacetate, their ability to form acetoacetatewas also tested (Table 2). No acetoacetate arosefrom ,3-hydroxy-,3-methylglutarate, whereas bothmevaldate and f3-hydroxy-f3-methylglutaryl-CoAwere rapidly and efficiently converted to aceto-acetate. Indeed, considerably more acetoacetatewas formed from mevaldate and ,B-hydroxy-,8-methylglutaryl-CoA than from mevalonate. Al-though less mevalonate was present, that remain-ing after 30 min of incubation was well above theKm concentration for this enzyme system (9);
thus, acetoacetate formation from mevalonateproceeded at close to maximal velocity. Bothmevaldate and f3-hydroxy-f3-methylglutaryl-CoAtherefore appeared to be likely candidates forintermediates between mevalonate and aceto-acetate. In each case, acetoacetate formation wastotally dependent on the presence of both enzymeand substrate, and omission of either NAD orCoA reduced the yield of acetoacetate from meva-lonate by over 85 %. NADP did not replace NAD.
Despite the ability of enzyme preparationsfrom mevalonate-grown cells to convert meval-date to acetoacetate, the data of Table 3 rule outparticipation of mevaldate as a free intermediatebetween mevalonate and acetoacetate. In the pres-ence of a mevaldate pool, little or no labeling ofreisolated mevaldate was observed despite sub-stantial incorporation of isotope from mevalonateinto acetoacetate. The molar ratios of mevaldateto mevalonate were 12.5 and 69 in trials II andIII, respectively. Decreased total isotope incor-poration into acetoacetate was observed on in-creasing the size of the mevaldate pool, althoughthe extent of dilution was far less than anticipatedwere mevaldate a free intermediate. The observeddecrease is readily explained as a consequence ofcompetitive inhibition of mevalonate oxidationby mevaldate, which has the correct structure for
TABLE 4. Effect of carrier ft-hydroxy-fl-methyl-glutaryl-CoA on the extent of incorporation of
isotope from mevalonate-2-C14 intoacetoacetatea
j5-Hydroxy-jS- Acetoacetate Total radioactivitymethyl glutaryl- formed found in acetoacetatebeoA
umoles ;umoles
None 0.14 1.7 X 1051.56 0.65 1.0 X 1063.10 0.82 4.7 X 104
a The following were incubated at 30 C in 1.3 ml:100 ,moles of Tris chloride, pH 8.0; 2.7 ,umoles ofCoA; 4.0 jAmoles of NAD; 0.38 ,Amole of DL-meva-lonate-2-CQ4 (1.15 X 106 counts per min per,umole);2.0 mg of dialyzed crude extract protein; and,B-hydroxy-ft-methylglutaryl-CoA free from ft-hy-droxy-,B-methylglutaric acid as indicated. After100 min, reaction mixtures were chilled and de-proteinized with 25% trichloroacetic acid. Samplesof the protein-free supernatant liquid wereanalyzed chemically for acetoacetate. To twoother samples, 54.5 ,umoles of carrier acetone wereadded. Acetone 2,4-dinitrophenylhydrazone wasthen prepared, purified to constant specific actiVityby paper chromatography, eluted, and counted.bCounted as acetone 2,4-dinitrophenylhy-
a competitive inhibitor of mevalonate:NAD oxi-doreductase (acylating CoA) (9).By contrast, even a fourfold molar ratio of car-
rier f3-hydroxy-f-methylglutaryl-CoA to meva-lonate significantly decreases incorporation ofisotope from mevalonate into acetoacetate (Table4). Three interpretations are that 3-hydroxy-,B-methylglutaryl-CoA is a free intermediate, thatit may act solely as a competitive inhibitor formevalonate oxidation [3-hydroxy-3-methylglu-tarate competitively inhibits mevalonate: NADoxidoreductase (acylating CoA) (9)], or thatboth effects operate together. That isotopeincorporation was depressed far more effectivelyby fl-hydroxy-3-methylglutaryl-CoA than bymevaldate suggests that competitive inhibitionalone may be an insufficient explanation, since allknown mevalonate-analogue competitive inhibi-tors have about the same K4 value (9). Therefore,more direct evidence was sought that,-hydroxy-3-methylglutaryl-CoA was an intermediate.Isolation of f-hydroxy-3-methylglutaryl-CoA.
After several unsuccessful attemps to detect inter-mediates between mevalonate and acetoacetate,a NAD-regenerating system (pyruvate plus lactatedehydrogenase) was added to shift the overallequilibrium in favor of product formation. Pyr-uvate disappearance, which was strictly depend-ent on the presence of the bacterial enzyme, wasused to follow the course of the reaction. Thefollowing were incubated at 30 C for 18 hr in2.0 ml: 100 ,moles of potassium phosphate, pH7.0; 10 pmoles of CoA; 16 ,umoles of reducedglutathione; 1.1 ,moles of NAD; 12 ,umoles ofsodium pyruvate; 1.1 ,umoles of DL-mevalonate;2,000 enzyme units of crystalline L-lactate:NADoxidoreductase; and 1.0 mg of dialyzed 25 to 50%(NH4)2SO4 fraction protein. Bacterial enzymewas omitted from the control incubation mixture.The reaction mixtures were then brought to pH5.0 with HCl, lyophilized, dissolved in water, andchromatographed in 95% ethyl alcohol-0.1 M
sodium acetate (v/v) and in isobutyric acid-15N NH40H-EDTA-water (62:12:1:28) (16).Compounds were visualized with nitroprussidereagent both before and after alkali treatment. Aspot visible after alkali treatment but absent fromcontrol incubations had an RF in both solventsidentical to that of ,-hydroxy-,B-methylglutaryl-CoA (0.54 and 0.70 in the first and secondsolvents, respectively). Further evidence for for-mation of f3-hydroxy-3-methylglutaryl-CoA was
provided by isolation of radioactive f3-hydroxy-,3-methylglutarate formed from mevalonate-2-C'4in a comparable incubation mixture (Table 5).Enzyme activities present in cell-free extracts.
Cell-free extracts of mevalonate-grown cells
TABLE 5. Incorporation ofisotope from mevaloniate-2-C14 into,-hydroxy-,s-methylglutaratea
Incubation mixture in HMG fractionbv Incorporationc
Complete....... 1.2 X 105 35No enzyme ...... 7.2 X 10O 2.2
a The following were incubated at 30 C in 4.1 ml:100 ,moles of potassium phosphate, pH 7.0; 11,umoles of CoA; 16 ,moles of reduced glutathione;1.2 /umoles of NAD, 20,umoles of sodium pyruvate;8.0 ,umoles of DL-mevalonate-2-C'4 (specific ac-tivity = 4.2 X 104 counts per min per ,umole);4,000 enzyme units of crystalline L-lactate:NADoxidoreductase; and 5.5 mg of the dialyzed 25to 50% (NH4)2S04 fraction protein. Bacterialenzyme was omitted in the control. After 42 hr,samples of each reaction mixture equivalent to1.65 X 104 initial counts per min were made 1 Nin KOH and were incubated for 30 min at roomtemperature to hydrolyze bound forms of ,B-hydroxy-,3-methylglutarate. The pH was adjustedto 3.0 with H2S04, solvent was removed in an airstream, and the dry residue was extracted with4 ml of absolute ethyl alcohol. The ethyl al-cohol extract was dried, dissolved in 0.6 ml of3 N H2SO4, mixed with 2 g of Celite, and trans-ferred to a Celite column (32 by 1.9 cm) equil-ibrated with CHC13 (saturated with 3 N H2SO4).Fractions were eluted first with 200 ml of 100%CHC13, then with 500 ml of 85% CHCl3-15% n-butanol (v/v). j3-Hydroxy-,8-methylgluraric acidfree from mevalonic acid was eluted at 400 to460 ml of the CHCI3-butanol fraction. Its identitywas established by coincidence of the radioactivepeak with that of the titratable peak for known,3-hydroxy-,3-methylglutarate from a third column.
b Expressed as counts per minute for the entireincubation mixture.
c Expressed as per cent of total (DL) mevalonate.
showed activity for the mevalonate:NAD oxi-doreductase (acylating CoA), the mevaldate:NAD oxidoreductase (acylating CoA), the meva-lonate:NAD oxidoreductase, and the NADH2oxidoreductase reactions (Table 6). The furtherpurification, kinetic properties, and competitiveinhibition of mevalonate:NAD oxidoreductase(acylating CoA) have been reported elsewhere(9). fl-Hydroxy-3-methylglutaryl-CoA lyase ac-tivity was detected as described by Bachhawatet al. (1) by incubating 1.8 mg of 25 to 50%(NH4)2SO4 fraction protein, 200 ,umoles of potas-sium phosphate (pH 7.0), 16 MAmoles of reducedglutathione, 10 Amoles of MgC12, and 1.2 ,umolesof #3-hydroxy-f-methylglutaryl-CoA for 1 hr at30 C in 1.0 ml. Samples were then analyzed di-rectly for acetoacetate. The complete systemformed 194 m,umoles of acetoacetate. Controlsminus enzyme or minus f3-hydroxy-f3-methylglu-
taryl-CoA formed 35 and 18 m,umoles of aceto-acetate, respectively.# - Hydroxy - , - methylglutaryl - CoA hydro-
lase was detected as described by Dekker et al.(6) by incubating the following at 30 C in 1.0 ml:200 ,umoles of potassium phosphate (pH 7.0), 20,umoles of EDTA, 2.3 ,umoles of (-hydroxy-fl-methylglutaryl-CoA, and 0.90 mg of dialyzed25 to 50% (NH4)2SO4 fraction protein. After 1hr, 1.0 ml of 9% metaphosphoric acid was added,precipitated protein was removed by centrifuga-tion, and a sample was analyzed for free sulfhy-dryl groups (22). The complete system formed230 m,moles of free sulfhydryl compounds. Con-trols minus 13-hydroxy-fl-methylglutaryl-CoA orminus enzyme gave no detectable color.
DIscussIoNThe pathway proposed for catabolism of meva-
lonate in actinomycete S4 is given in Fig. 1.Conversion of mevalonate to acetoacetate in-
volves formation of free fl-hydroxy-13-methylglu-taryl-CoA, but not of free mevaldate. The failureto find free mevaldate as an intermediate parallelsthe observations of Durr and Rudney using yeastfl-hydroxy-#-methylglutaryl-CoA reductase (7).Salient features are that the S4 enzymes all aresoluble, that NAD rather than NADP is used asoxidant, and that the reactions favor mevalonateoxidation. Yeast and liver enzymes apparentlycatalyze essentially irreversibly the reducednicotinamide adenine dinucleotide phosphate(NADPH2)-dependent reduction of #-hydroxy-
TABLE 6. Preliminary fractionation of crude cell extracta
Specific Recov- Specific Recov- Specific R ScoveSpecif cactivity' ery5 activity ery activity activity ver
Crude extract ...................... 5.7 10085,000 X g supernatant liquid........ 7.2 100 2.8 100 21 100 0 085,000 X g pellet..................... 0 0 0 0 0 0 6.4 12325 to 50% (NH4)2SO4 fraction........ 43.5 80 16.4 60 75 38 0 0
Oxidoreductase activities were assayed at 30 C in 1.0 ml by the absorbance change at 340 m; due tothe appearance or disappearance of NADH2. The reactions were started by addition of enzyme.
b The assay cuvette contained 70 ,umoles of potassium phosphate, pH 7.0; 1.0 ,mole of CoA; 4.0 ,molesof reduced glutathione; 5.0 ,umoles of MgSO4; 1.5 ,umoles of NAD; and 12 ,Amoles of potassium DL-mevalonate, pH 7.0.
c The assay cuvette contained the additions given in footnote b, except that mevalonate was replacedby 10 jmoles of freshly prepared potassium DL-mevaldate.
d The assay cuvette contained the additions given in footnote b, except that CoA was omitted andNAD was replaced by 0.3 umole of NADH2 (5).
e The assay cuvette contained 70 ,Amoles of potassium phosphate, pH 7.0, and 0.3 j"mole of NADH2.f Expressed as milliunits per milligram of protein. One oxidoreductase milliunit is that amount of
enzyme catalyzing the reduction of 1 m,umole of NAD per min (International Union of Biochemistry,Report of the Commission on Enzymes, Pergamon Press, New York).
g Except for NADH2 oxidoreductase, recoveries are based on the activity of the 85,000 X g fraction,since the NADH2 oxidoreductase activity of the crude extract precluded accurate measurement of otheroxidoreductase activities.
H3C OH
HO-CH2 COOH
2NAD 2NADH2 + 2H+
CoA-SH
MEVALONATE
H3C OH CH3
CoA-S-- COOH * H2A ~~~~OOH
HMG-CoA CH3-C-S-CoA ACETOACETATE
ACETYL-CoAFIG. 1. Proposed pathway for catabolism ofmevalonate in actinomycete S4.
f-methylglutaryl-CoA to mevalonate (7). Thisapparent contradiction no doubt results from thepresence in S4 extracts of a 3-hydroxy-3-meth-ylglutaryl-CoA hydrolase and, more particu-larly, of a f-hydroxy-f3-methylglutaryl-CoA:acetoacetate lyase. Thus, the overall reactionshould favor mevalonate oxidation.The ability to grow on citric acid cycle inter-
mediates or on acetate and the excretion into themedium of small quantities of a-ketoglutarateimply functioning tricarboxylic acid and glyoxy-late cycles. The organism thus may be provision-ally considered to operate on a "two-carboneconomy." Presumably, acetoacetate is furthermetabolized to acetyl-CoA, which enters the tri-carboxylic acid and glyoxylate cycles.
ACKNOWLEDGMENTS
This investigation was supported by Public HealthService grant GM-06468 from the Division of GeneralMedical Sciences.
LITERATURE CITED
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