Activities of a-ketoisovalerate, pyruvate, and a-ketoglutarate dehydrogenases in a mutant of Bacillus subtilis

C. L. T u 3 AND TOSHI KANEDA Alberia RP.SC(I~C/ I C o i i t ~ ~ i l , E ( l n ~ o t ~ i o t ~ . A Ih~t . io

Accepted December 31, 1975

Tu. C. L.. and T. KANEDA. 1976. Activities of a-ketoisovalerate, pyruvate, and a-ketoglutarate dehydrogenases in a mutant of Bncillrrs siihiilis. Can. J . Microbiol. 22: 592-597.

An acetate-requiring leaky mutant was induced from Bncillus subiilis 168, and activities of its three a-keto acid dehydrogenases were compared with the respective activities of the parent strain. Both pyruvate and a-ketoisovalerate dehydrogenase activities in the mutant were consid- erably lower, being only 10-17% of those of the parent, but a-ketoglutarate dehydrogenase activity was unchanged. These dehydrogenases are complexes composed of three enzymes: a carboxylase, a lipoic reductase-transacylase, and a dihydrolipoyl dehydrogenase. The carbox- ylase activity of the affected complexes was no different. Total dihydrolipoyl dehydrogenase activity was only one-third. Thus dihydrolipoyl dehydrogenase is the defective enzyme in the twodehydrogenaae complexes: theactivity remainingin themutant isaccountedforby theactivity of the intact a-ketoglutarate dehydrogenase.

Tu. C. L., et T . KANEDA. 1976. Activities of a-ketoisovalerate, pyruvate and a-ketoglutarate dehydrogenases in a mutant of B~cillris sirhiilis. Can. J . Microbiol. 22: 592-597.

Un "leaky" mutant exigeant de ['acetate a etb induit chezBoc~illr~s sri1~iili.s 168 et I'activite de ses trois a-cCto-acides deshydrogenases a ete comparee a I'activitt de la souche parent. L'activitC de la pyruvate deshydrogenase et de I'a-cetoisoval6rate dCshydrogCnase chez le mutant est beaucoup plus faible n'ayant que 10-17% de I'activite de la souche parent; toutefois, I'activiti de I'a-cetoglutarate demeure inchangee. Ces deshydrogCnases sont des complexes composees de trois enzymes: une carboxylase, une lipoi'que rkductase transacylase et une dihydrolipoyl dCshydrogCnase. L'activite de la carboxylase des complexes alteres est la mkme. L'activite totale de la dihydrolipoyl deshydrogenase n'est que du tiers de I'activite de la souche parent. Ainsi la dihydrolipoyl deshydrogenase est I'enzyme difectueux chez les deux deshy- drogknases du complexe; I'activite restante chez le mutant est due i I'activite de I'a-cCtoglu- tarate deshydrogknase intacte.

[Traduit par le journal]

Namba et al. have shown that three different a-keto acid dehydrogenases are present in Bacill~rs subtilis (9). Two of them, the pyruvate and a-ketoglutarate dehydrogenases, have enzy- matic characteristics essentially identical with those of similar enzymes found in Escherichia coli (7). The third one is a new a-keto acid dehydrogenase that is absent from E. coli and is capable of catalysing oxidative decarboxylation of a-keto acids related to valine, isoleucine, and leucine in a way similar to that of the two other enzymes. All three dehydrogenases are multi- enzyme complexes with molecular weights of millions. The sequences of reactions catalysed by these complexes may be represented by the scheme shown in Fig. 1.

R - C - C O O H r 1PP.EI - C 0 2 ' R - C H - T P P . E l (1) I, I 0 O H

FADrcd . E3 + NAD' - FADo, . i 3 t N A D H + H ' (5)

FIG. 1. A sequence of generalized reactions catalysed by three a-keto acid dehydrogenase complexes. El represents carboxylase; E2, lipoic reductase-transacylase; and E3, dihydrolipoyl dehydrogenase. CH3-, (CH,),-CHz- CH, and HOOC-CH,-CH, for R are pyruvic, a-ketoiso- valeric, and a-ketoglutaric acids, respectively. Abbrevia- tions used are TPP, thiaminepyrophosphate; HS-CoA, coenzyme A ; FAD,, and FAD,,,, oxidized and reduced forms of flavine adenine dinucleotide; NAD, nicotin- amide adenine dinucleotide.

'Received July 18, 1975. Reactions 1, 2, and 3 are catalysed by enzymes 2Contribution No. 756 from the Alberta Research

Council, Edmonton, Alberta. E l and E2 and include the appropriate a-keto 3Alberta Research Council Postdoctoral Fellow 11970- acid substrate. However, reactions 4 and 5 9

1971). catalysed by enzyme E3, involve no substrate.

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Thus it seems likely that whereas El and E2 are specific to their respective complexes, E3 is non- specific and may well be common to two, or perhaps all three, complexes. This possibility has been examined by use of a mutant having almost no pyruvate dehydrogenase activity, and the results are presented here.

Materials and Methods Microorganisrns

The parental strain from which mutants were derived is Bacillus srrbtilis 168 trp- kindly supplied by Dr. S. H. Taubman. Acetate mutants were induced from the strain using ethyl methanesulfonate as the mutagen and were enriched by selection in staphcillin, according to the procedure of Freese and Fortnagel (2).

P~.eparation of Cell Extracts A litre of medium prepared from 6 g of Trypticase Soy

Broth Powder (Baltimore Biological Laboratories) (one- fifth normal strength) and inorganic salts (4) was placed in a Fernbach flask, inoculated with the organism, and incubated 16 h at 30 'C on a rotaryshaker. The cells were then harvested, washed once with 0.85% NaCl solution, and suspended in 1.5 volumes of 10 m M phosphate buffer (pH 7.0). A cell-free extract was prepared from the suspension by treating with ultrasonic oscillation as reported (6). In the preparation of cell-free extracts, special care was taken to maintain the reproducibility of specific activities of the three a-keto acid dehydrogenases. Conditions were kept as nearly identical as possible and the preparation of the extracts was done by the same person throughout the work (40 ml of the suspension in a 100-ml beaker was treated by a +-in. tip of Biosonik BP-111 ultrasonic system (Bronwill Scientific Co., Rochester, New York) dipped in 15 mm below the water surface).

Partially purified preparation was prepared from the crude extract by ammonium sulfate fractionation (4&60% saturation) as before (9).

Enzywie Assays The assay scheme is shown in Fig. 2. Decarboxylating

activity, as measured by production of 14COz from an appropriate [I-14C]a-keto acid substrate, was independent of the presence of nicotinamide adenine dinucleotide (NAD) and coenzyme A (CoA); dehydrogenase activity, similarly measured, was dependent on the presence of NAD and CoA. A correction for nonenzymatic decar- boxylation was made by running an assay in which the native enzyme was replaced with boiled enzyme.

The reaction mixture for the dehydrogenase assay

FIG. 2. Assay scheme for activities of decarboxylase, dehydrogenase in whole, and carboxylase alone.

ACID

consisted of the following components (in pmol) in a total volume of 0.3 ml : NAD, 0.15; CoA, 0.15; thiaminepyro- phosphate (TPP), 0.15; MgCI2, 1; KHz-K2HP04 (pH 7.0), 40; mercaptoethanol, 0.1; [I-14C]pyruvate (13 000 cpm/pmol), or [I-'4C]a-ketoglut3rate (16 000 cpm/pmol), or [l-14C]a-ketoisovalerate (14 500 cpmlpmol), 1 ; and sufficient cell-free extract to give 0.1-0.5 mg pro- tein for pyruvate or 0.5-2.5 mg for a-ketoglutarate and a-ketoisovalerate. In these ranges, enzyme activity is proportional to the amount of enzyme protein. Each assay was run with several different amounts of enzyme protein in the range of proportionality to obtain a mean value.

The cell-free extracts used as the enzyme source con- tained active NADH oxidase (EC 1.6.2.1) and acyl CoA hydrolase (EC 3.1.2.1) (9). Thus the reaction products NADH and acyl CoA were continuously removed from the system to regenerate NAD and CoA, and the com- pletion of reactions 1 to 5 required only small amounts of NAD and CoA.

Carboxylase activity was measured under similar conditions except that the NAD and CoA were replaced by K3Fe(CN)6 (10 pmol or otherwise specified).

The reaction mixture was placed in a test tube fitted with a rubber cap through which a coiled wire had been pushed to hold a petti-cup containing 0.2 ml of 1 M hyamine hydroxide in methanol (New England Nuclear, Boston, Massachusetts) halfway down the tube. The reaction was carried out at 30 "C for 20 min when pyru- vate was the substrate, 30 min for a-ketoglutarate, and 40 min for a-ketoisovalerate, then stopped with the addi- tion of 0.2 ml I M HC104 to the reaction mixture by means of a syringe through the rubber cap. The test tube was then shaken 30 min to allow complete absorption of L4C02 , and the petti-cup transferred to a scintillation counting vial. Ten rnillilitres of a toluene-based scintilla- tion mixture (Liquifluor, New England Nuclear) was added to the vial and the radioactivity was measured in a Beckman Liquid Scintillation Counter.

DEHYDROGENASE

Dihydrolipoyl Deliydrogenase Assay The enzyme was assayed by measuring the disappear-

ance of the sulfhydryl group from dihydrolipoic acid in the presence of catalytic amounts of NAD, lactic acid dehydrogenase, and pyruvate. The remaining sulfhydryl group was measured calorimetrically by reacting it with 5,5'-dithio bis-Znitrobenzoic acid (DTNB) (1).

The reaction mixture, in a total of 0.25 ml, contained (in pmol) dihydrolipoic acid, 1.5 or otherwise specified; NAD, 0.25; pyruvate, 5; phosphate buffer (pH 8.0), 50; lactic acid dehydrogenase 0.5-1.0 unit; 0.1-0.5 mg protein of the dialysed crude cell-free extract. The reaction was carried out at 30 "C for 15 min or as specified and stopped by addition of 0.5 ml 0.4% DTNB in 0.05 M phosphate buffer (pH 8.0), whereupon the solution turned yellow rapidly (1 1 min). The yellow solution was diluted to a suitable concentration and the optical density was measured at 412 nrn (E, 13 600 M-I cm-' for DTNB). /." Chemicals

[l-14C]a-ketoisovalerate was prepared enzymatically from L-[l-14C]valine according to Meister (8). [l-14C]- pyruvate (Na salt) was crystallized from a water-acetone mixture to remove pyruvate polymers, whereas [1-14C]u- ketoglutarate was used as received. These 14C-substrates

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CAN. 1. MICROBIOL. VOL. 22, 1976

TABLE 1. Activities of a-keto acid dehydrogenases and decarboxylation in crude extracts of strains of Bacillris subtilis

a-Ketoisovalerate" Pyruvate" a-Ketoglutarate"

Prepn. Dehydrog- Decarboxyl- Dehydrog- Decarboxyl- Dehydrog- Decarboxyl- Strain No. enase ation enase ation enase ation

Mutant 1 14 30 - 0 10 320 240 44 2 20 18 - 0 4 210 303 24 3 17 8 - 0 3 490 528 47

Parent 1 120 86 1000 5 280 146 74 2 120 3 1320 2 000 371 40

"Activity is expressed in nmol CO1 produced/h rng protein.

were purchased from the New England Nuclear Corp. Coenzyme A, NAD, dihydrolipoic acid, lactic acid dehydrogenase, and DTNB were obtained from Sigma Chemical Co., St. Louis, Missouri.

Results Isolation of Acetate Mutants and their Growth

CI7aracteristics From primary platings of cultures treated with

the mutagen, 5974 colonies were picked. Further selection, based on the response to acetate, reduced these to 60 isolates, all of which were found to be leaky mutants. Among them, mutant B2A was chosen for the present study, since it showed a 3-day lag period, longer than any of the other isolates, before active growth took place in Spizizen's minimal liquid medium (10). When acetate was added to the medium, the organism initiated growth immediately. Glutamate was found to stimulate growth. Mutant B2A loses viability very rapidly, usually within 3 days. Although it does not sporulate, a sporulating medium, AK-medium (Difco), greatly reduced the loss of viability of mutant B2A.

Activities of a-Keto Acid Dehydrogenases and a-Keto Acid Decarboxylase

The specific activities of the enzymes in extracts of the parent and mutant strains are shown in Table 1. We were forced to use crude extracts for these experiments since a-keto- glutarate dehydrogenase was, as reported pre- viously (9), very unstable and its activity dropped rapidly so that it had to be measured as soon as the extract had been prepared. Never- theless, the following general conclusions can be drawn. The activity of a-ketoisovalerate dehydro- genase in the mutant was only 12-17% of that present in the parent, and the activity of pyruvate dehydrogenase was hardly detectable. Activities

of a-ketoglutarate dehydrogenase in the two strains, however, were not much different.

Because an extremely large activity was found for pyruvate decarboxylation, the nature of the reaction was further investigated. The reaction product of the decarboxylation was identified as acetoin by its color reaction (5) and the entire decarboxylation could be accounted for by the following reaction :

This enzyme, however, was absent in the partially purified extract (Table 4). Acetoin has been found to be a major product in the culture medium of B. subtilis (5).

Carboxylase Activities of a-Ketoisovalerate Dehy- drogenase in the Parent and Mutant Strains

Namba et al. (9) have shown that the car-

FIG. 3. Carboxylase activity as a function offerricyanide concentration.

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TU AND KANEDA

FIG. 4. Activities of dihydrolipoyl dehydrogenase of the parent strain as a function of incubation time (A), amount of enzyme protein (B), and substrate concentration (C).

TABLE 2. Carboxylase activity of a-ketoisovalerate dehydrogenase and dihydrolipoyl dehydrogenase activity in crude and dialysed extracts of strains of Bacillrts subrilis

a-Ketoisovalerate Dihydrolipoyl

Prepn. Dehydrogenase4 Carboxylase" dehydrogenaseb Strain No. (crude extract) (crude extract) (dialysed extract)

Mutant 1 2 3

Parent 1 2 3 4

OActivity is expressed in nmoi C 0 2 produced/h rng protein (crude extract). bActivity is expressed in nmol SH oxidizedlh mg protein (dialysed extract) <Number o f different preparations used.

boxylase activity of cr-ketoisovalerate dehy- drogenase, similar to that of pyruvate dehydroge- nase (7), can be assayed independently from other activities by the use of ferricyanide as the electron acceptor. Figure 3 shows the carboxylase activity as a function of K,Fe(CN), concentra- tions. K,,, value for K,Fe(CN), was calculated to be 9 mM. A K,Fe(CN), concentration of 30 mM was used routinely for the assay of carboxylase activity.

The activities of the whole dehydrogenase re- action and the carboxylase reaction alone in extracts of the mutant and parent are shown in Table 2. The dehydrogenase activity of the

mutant was only 10% of that found in the parent, whereas the carboxylase activity of the mutant was as great as or, in many cases, greater than, that of the parent. This indicates that in the mutant the synthesis of the El of Fig. 1 is not affected at all, but either E2 or E3, or both, are defective.

Dihydrolipoyl Dehydrogenase The assay conditions for dihydrolipoyl dehy-

drogenase were established by the following observations. The activity in the parent strain is proportional to the amount of enzyme protein up to 0.50 mg per assay and linear for at least 15 min

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596 CAN. J. MICROBIOL. VOL. 22, 1976

TABLE 3. Activities (nmol CO, produced per hour) of a-ketoisovalerate dehydrogenase in purified extracts of the mutant and parent measured separately or together

- -

Enzyme sources (0.5 mg each)

Parent + mutant

Supplements Parent Mutant Observed Calcd.

(a) K3Fe(CN)6 104 1 44 242 (b) NAD, CoA 120 40 198 (c) None 30 30 46

Dehydrogenase (6-c) 90 10 152 100 Carboxylase (a-c) 74 114 196 188

TABLE 4. Activities (nmol CO, produced per hour) of pyruvate dehydrogenase in purified extracts of the mutant and parent measured separately or together

Enzyme sources (0.2 mg each)

Parent + mutant

Supplement Parent Mutant Observed Calcd.

(a) K3Fe(CN)6 96 8 1 177 (6) NAD, CoA 432 54 813 (c) None 66 46 117

Dehydrogenase (6-c) 366 6 696 372 Carboxylase (a-c) 30 3 3 60 63

(Fig. 4A, B). The concentration of dihydrolipoic dehydrogenase was observed by the addition of acid at 4 m M was sufficient to give the maximum an animal dihydrolipoyl dehydrogenase. The two rate of dehydrogenation (Fig. 4C). Table 2 shows systems do not couple. the specific activity of dihydrolipoyl dehy- drogenase of the mutant and parent strains Discussion measured under the established assay conditions. The observations reported here show that the The activity of the mutant was only one-third following biosynthetic relationships should exist that of the parent. among the three a-keto acid dehydrogenases in

Coupling Between the Parent and Mutant En- zymes

A possible coupling between the defective a-ketoisovalerate dehydrogenase of the mutant and the complete enzyme of the parent was investigated. The measured activity of a mixture of equal amounts of parent and mutant extracts was about the same as the sum of the activities of each type measured separately (Table 3). When the pyruvate dehydrogenases were assayed in this manner, however, the mixture produced an activity nearly double the sum of the activities of the individual enzymes (Table 4).

. -

B. subtilis. Dihydrolipoyl dehydrogenase, E3 in reactions 4 and 5, is the enzyme defective in, o r missing from, the complex of both the pyruvate and a-ketoisovalerate dehydrogenases synthe- sized by the mutant. Enzyme E3 of cc-ketoglu- tarate dehydrogenase is intact in the mutant. I t has been reported that, in another mutant, both pyruvate and a-ketoglutarate dehydrogenase are defective. A citrate-requiring mutant of B. subtilis, cit 22, lacks both the dehydrogenase activities as a result of the absence of dihydroli- poyl dehydrogenase activity (3). No statement was given on a-ketoisovalerate dehydrogenase.

At present it is not known whether the syn- Possible Interaction of the Mutant Enzymes with thesis of E3 of the pyruvate dehydrogenase and

an Animal Dihydrolipoyl Dehydrogenase the a-ketoisovalerate dehydrogenase in strain No change in the activity of a-ketoisovalerate B,A is controlled by the same gene or by two

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TU AND KANEDA 597

separate ones. Many attempts were made to determine whether the mutant is the result of a single mutation or multi-mutations. Determining the frequency of reversion (2) was found t o be difficult because the mutant could grow without acetate after a lag period. Colonies of revertants and mutants were difficult to distinguish on agar plates although several growth media were examined.

The present observations show that enzyme E3 of the complete a-ketoglutarate dehydrogenase does not couple with either the defective a- ketoisovalerate dehydrogenase or the defective pyruvate dehydrogenase. Enzyme E3 of the complete a-ketoisovalerate dehydrogenase does not couple with the defective a-ketoisovalerate dehydrogenase either. This is not so in the case of pyruvate dehydrogenase, where enzyme E3 of the complete complex does couple with the defective complex to restore its activity. Thus enzymes E2 and E3 of the pyruvate dehydrogenase are likely exposed on the outside of the dehydrogenase complex, and thus able to interact with each other.

It is not presently known whether the defective dehydrogenases totally lack enzyme E3 or whether an inactive E3 peptide remains in the complexes as a constituent to maintain the struc- ture. The fact that the a-ketoisovalerate dehv- drogenase and the pyruvate dehydrogenase fro; the mutant both retained partial activities (10- 17%) suggests a genetically modified E3 attached to the respective dehydrogenase complex. This is known to be the case for tryptophan synthase (EC 4.2.1.20) of E. coli (1 1). Although, as reported earlier (9), the three dehydrogenases, particularly a-ketoisovalerate dehydrogenase and pyruvate dehydrogenase, are rather difficult to separate, newer techniques are being investi- gated to isolate each dehydrogenase in pure form and thus facilitate differentiation of the two possibilities.

The present results provide the basis for a more extensive study by genetic means of the control of the enzyme E3, which is a component of the three dehydrogenase complexes.

Acknowledgment

This investigation was supported by grant MT-1660 of the Medical Research Council, Ottawa, Canada.

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6. KANEDA, T . 1973. Biosynthesis of branched long- chain fatty acids from the related short-chain a-keto acid substrates by a cell-free system of Brrci!llrs slrbtilis. Can. J. Microbiol. 19: 87-96.

7. KOIKE, M. , L. J . REED, and W. R . CARROLL. 1960. a-Keto acid dehydrogenation complexes. I. Purifi- cation and properties of pyruvate and a-ketogluta- rate dehydrogenation complexes of Eschcrichirr coli. J . Biol. Chem. 235: 1924-1930.

8. MEISTER, A. 1952. Enzymatic preparation of a-keto acids. J . Biol. Chem. 197: 309-317.

9. NAMBA, Y., K. YOSHIZAWA, A. EJIMA, T . HAYASHI, and T. KANEDA. 1969. Coenzyme A- and nico- tinamide adenine dinucleotide-dependent branched chain a-keto acid dehydrogenase. J . Biol. Chem. 244: 4437-4447.

10. SPIZIZEN. J . 1958. Transformation of biochemically deficient strains of Bnc~illris srrbtilis by deoxyribonu- cleate. Proc. Natl. Acad. Sci. U.S.A. 44: 1072-1078.

11. YANOFSKY, C., J . ITO, and V. HORN. 1966. Amino acid replacements and genetic code. Cold Spring Har- bor Symp. Quant. Biol. 31: 151-162.

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