Vol. 148, No. 2JOURNAL OF BACTERIOLOGY, Nov. 1981, p. 639-6460021-9193/81/110639-08$02.00/0

Isolation of a Specific Lipoamide Dehydrogenase for aBranched-Chain Keto Acid Dehydrogenase from

Pseudomonas putidaJOHN R. SOKATCH,* VICKI McCULLY, JANET GEBROSKY, AND DAVID J. SOKATCHDepartment ofMicrobiology and Immunology, University of Oklahoma Health Sciences Center,

Oklahoma City, Oklahoma 73190

Received 20 April 1981/Accepted 10 July 1981

We purified lipoamide dehydrogenase from cells ofPseudomonasputida PpG2grown on glucose (LPD-glu) and lipoamide dehydrogenase from cells grown onvaline (LPD-val), which contained branched-chain keto acid dehydrogenase.LPD-glu had a molecular weight of 56,000 as determined by sodium dodecylsulfate-polyacrylamide gel electrophoresis, and LPD-val had a molecular weightof 49,000. The pH optimum for LPD-glu for reduced nicotinamide adeninedinucleotide oxidation was 7.4, compared with pH 6.5 for LPD-val. When oxidizednicotinamide adenine dinucleotide was included in the assay mixture, the pHoptima were 7.1 and 5.7, respectively. There was also a difference in pH optimabetween the two enzymes for oxidized nicotinamide adenine dinucleotide reduc-tion, but the Michaelis constants and maximum velocities were similar. A purifiedpreparation of branched-chain keto acid dehydrogenase, which was deficient inlipoamide dehydrogenase, was stimulated 10-fold by LPD-val but not by LPD-glu, which suggested that the branched-chain keto acid dehydrogenase of P.putida has a specific requirement for LPD-val. In contrast, a partially purifiedpreparation of 2-ketoglutarate dehydrogenase that was deficient in lipoamidedehydrogenase was stimulated by LPD-glu but not by LPD-val, indicating thatthis complex has a specific requirement of LPD-glu.

The most important function of lipoamidedehydrogenase (EC 1.6.4.3) is that it serves as asubunit of pyruvate dehydrogenase and 2-keto-glutarate dehydrogenase (18). These multien-zyme complexes from Escherichia coli consistof three subunits with analogous functions. Sub-unit El is the decarboxylase-dehydrogenase,subunit E2 is the transac'ylase and is the subunitwhich contains covalently bound lipoic acid, andsubunit E3 is lipoamide dehydrogenase and cat-alyzes the following reaction:

E2-lip(SH)2 + E3 E2-lipS + E3-H2

E3-H2 + NAD+ E3 + NADH + H+

The mammalian pyruvate dehydrogenase com-plex contains these three subunits plus a kinaseand a phosphatase, which regulate enzyme ac-tivity by phosphorylation and dephosphoryla-tion (3). These and other functions of lipoamidedehydrogenase have been reviewed by Guest (5).

E. coli produces a single lipoamide dehydro-genase for both pyruvate dehydrogenase and 2-ketoglutarate dehydrogenase. Pettit and Reed(16) isolated lipoamide dehydrogenase from the

pyruvate and 2-ketoglutarate dehydrogenases ofE. coli and showed that the two proteins wereindistinguishable physically, chemically, andcatalytically. Later, Guest and Creaghan (6) andAlwine et al. (1) isolated mutants of E. coli K-12that had lesions in the structural gene for Ii-poamide dehydrogenase. Guest (4) mapped thelpd gene, which proved to be very close to aceEand aceF, the structural genes for subunits Eland E2 of pyruvate dehydrogenase. The genesfor subunits El and E2 of 2-keto-glutarate de-hydrogenase (sucA and sucB) were located 14min from aceE and aceF. Mutations in lpd wereaccompanied by a drastic reduction in lipoamidedehydrogenase, a dual requirement for acetateand succinate, and the loss of pyruvate and 2-ketoglutarate dehydrogenases. Mammals pro-duce several isozymes of lipoamide dehydroge-nase, but all have the same molecular weightand are thought to be conformational isomers(27).A study of the branched-chain keto acid de-

hydrogenase of Pseudomonas putida provedthat this enzyme was a multienzyme complexwhich had an E3 subunit similar to pyruvate

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640 SOKATCH ET AL.

and 2-ketoglutarate dehydrogenases (23). Dur-ing purification, the enzyme became deficient inlipoamide dehydrogenase. The purpose of thiswork was to determine whether there is a singlelipoamide dehydrogenase in P. putida for pyru-vate dehydrogenase, 2-ketoglutarate dehydro-genase, and branched-chain keto acid dehydro-genase or whether branched-chain keto acid de-hydrogenase has a specific lipoamide dehydro-genase. We purified lipoamide dehydrogenasefrom P. putida cells grown on glucose (LPD-glu), which contained pyruvate and 2-ketoglu-tarate dehydrogenases, and lipoamide dehydro-genase from P. putida cells grown on valinewhich contained all three keto acid dehydrogen-ases. The two lipoamide dehydrogenases haddifferent molecular weights and pH optima, andonly LPD-val stimulated branched-chain ketoacid dehydrogenase deficient in subunit E3.

MATERIALS AND METHODSOrganisms and growth conditions. P. putida

PpG2 was obtained originally from I. C. Gunsalus andwas grown as described previously (12). The syntheticmedium of Jacobson et al. (7) was used, with 0.3% L-valine and 0.05% L-isoleucine as the carbon sources.

Chemicals. NAD+, NADH, coenzyme A, and DL-lipoamide were obtained from Sigma Chemical Co.DL-Dlhydrolipoamide was prepared by the method ofReed et al. (19).Enzyme assays. Lipoamide dehydrogenase was

assayed during the purification procedure by themethod of Reed and Wilms (20). However, the pH ofthe assay mixture was 8.1, which was different fromthe pH optimum of either lipoamide dehydrogenaseisolated in this study; therefore, the specific activitiesreported below are low. Crude extracts contained largeamounts of alcohol dehydrogenase, which interferedwith the assay. For these assays, acetone was substi-tuted for alcohol as the solvent for DL-lipoamide. Oneunit of activity was defined as 1 ,umol of NADHoxidized per min per mg of protein. Branched-chainketo acid dehydrogenase was prepared as described inthe accompanying paper (23). The assays used forpyruvate, 2-ketoglutarate, and branched-chain ketoacid dehydrogenases were also as described in theaccompanying paper (23). Protein was determined byusing the Bio-Rad Laboratories protein assaiy.

Michaelis constants (Ki), mamum velocities(V.,.), and Hill coefficients were calculated by a pro-gram written in BASIC for the TRS-80 minicomputer(level II, with a 32K random access memory and a

single 5.25-inch [13.335-cm] disk drive). This programselected the best-fit lines for Lineweaver-Burk double-reciprocal plots (10) and for Hill coefficients (2) by theleast-squares method. This program also calculatedthe slopes and identified the intercepts. All kineticstudies were done at the pH optima listed below (seeTable 3). When the kinetics of NADH oxidation werestudied, the concentration of NAD+ was kept at 0.1mM, whereas the concentration of dihydrolipoamidewas varied. When the concentration of NAD+ was

varied, the concentration of dihydrolipoamide was

kept constant at 3.0 mM. When the kinetics of NAD+

J. BACTERIOL.

reduction were studied, the concentration of NAD+was varied and four concentrations of dihydrolipoam-ide were used (see Fig. 4 legend). The data in Fig. 4were plotted from duplicate determinations for eachpoint.

Purification of LPD-glu. The purification proce-dure shown in Table 1 started with 11 g of cells whichwere suspended in 56 ml of 50 mM potassium phos-phate buffer (pH 7.0) containing 1 mM EDTA and 0.5mM dithiothreitol (phosphate-EDTA-DTT buffer).The cell suspension was treated with sonic energy for12 min by using a Heat Systems model W140 sonicgenerator at a power setting of 7. The suspension ofbroken cells was centrifuged at 90,000 x g for 1 h toremove most of the NADH oxidase. The supernatantfraction from step 1 (56 ml) was centrifuged at 230,000x g for 3 h, which sedimented 71 U of dihydrolipoam-ide dehydrogenase but left 93 U in the supernatantfraction. Since virtually all of the pyruvate dehydro-genase and all of the 2-keto-glutarate dehydrogenasewere recovered in the pellet, the lipoamide dehydro-genase in the supernatant fraction was probably un-complexed.The pellet was dissolved in the minimum amount

of phosphate-EDTA-DTT buffer (final volume, 4.7ml), applied to a column of Sepharose CL-4B (2.5 by60 cm), and eluted with the same buffer. Fractions (4ml) were collected, and the contents of tubes 32through 52 were pooled. The pool from the SepharoseCL-4B column was heated at 650C for 15 min, whichinactivated the keto acid dehydrogenases, and thenthis pool was applied to a column of DEAE-SepharoseCL-6B (2.0 by 28 cm) equilibrated with phosphate-EDTA-DTT buffer. After the enzyme was loaded, thecolumn was washed with 100 ml of phosphate-EDTA-DTT buffer, and the protein was eluted with a lineargradient (250 ml of phosphate-EDTA-DTT buffercontaining 300 mM sodium chloride in the reservoirand 250 ml of phosphate-EDTA-DTT in the mixingchamber). Fractions (3 ml) were collected, and thecontents of tubes 68 through 96 were pooled. Theenzyme from step 5 (168 ml) was dialyzed overnightagainst two changes consisting of 2 liters of 25 mMpotassium phosphate buffer (pH 7.0) containing 1 mMEDTA and 0.5 mM dithiothreitol. The dialyzed en-

TABLE 1. Purification ofLPD-gluSpe-cific

Amt of Amt of actStep Fraction protein enzyme (pmnol/

(mg) (U) per mgof pro-tein)

1 90,000-x-g super- 641 207 0.32natant

2 230,000-x-g pellet 184 71 0.393 Sepharose CL-4B 40 49 1.2

column4 Heated enzyme 29 46 1.6

from step 35 DEAE-Sepharose 7.0 32 4.6

column6 Affi-Gel Blue col- 0.13 8.4 65

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zyme was applied to an Affi-Gel Blue column (2.3 by8 cm). This column had been equilibrated with 25 mMpotassium phosphate buffer (pH 7.0)-i mM EDTA-0.5 mM dithiothreitol and washed with 100 ml of thissame buffer. Protein was eluted by a linear sodiumchloride gradient, using 100 ml of 25 mM potassiumphosphate buffer (pH 7.0)-i mM EDTA-0.5 mM di-thiothreitol in the mixing chamber and 100 ml of thissame buffer containing 500mM sodium chloride in thereservoir. Lipoamide dehydrogenase usually appearedin the eluate near the end of the gradient (Fig. 1) andwas completely eluted with 170 ml of the same bufferused in the reservoir.

Purification of LPD-val. The purification proce-dure shown in Table 2 began with 8 g of cells anddiffered only in minor details from the purificationprocedure shown in Table 1. Virtually all of the li-poamide dehydrogenase and keto acid dehydrogenasecomplexes were sedimented by centrifugation at176,000 x g (Table 2, step 2), in contrast to thecorresponding step in Table 1, where much lipoamidedehydrogenase remained in the supernatant fraction.DEAE-Bio-Gel A was used in step 2 (Table 2) ratherthan DEAE-Sepharose, but the latter gave better res-olution and was used in subsequent preparations. Thepattern of elution of LPD-val from Affi-Gel Blue wasdifferent from the pattern obtained with LPD-glu (Fig.

7 .07

.6 .06

.5 .~~~~~~~~~~~~.05

4 ~~~~~~~~~~~~~~~~~.04ci) ~~~~~~~~~z

D.3 ~~~~~StartofSat o .03Gradient O c

.2 .02 LI

.1 -.01

-20 -10 0 100 20

ML

B ~~~~~~~~~~~~~~~~~.07

.3 .06

.05

-J.2 ~~~~~~~~~~~~~~~~.04* ~~~~~~~~~0.5MNaCI

_ ~~~start Of -.03zZ Gradient E

D 1.0

ML

FIG. 1. Chromatography ofLPD-glu (A) andLPD-val (B) on Affi-Gel Blue.

TABLE 2. Purification ofLPD-valSp act

Amt of Amt (umol/Step Fraction protein of en- min

(mg) zyme permg(m) (U) of pro-tein)

1 90,000-x-g superna- 648 76 0.12tant

2 176,000-x-g pellet 268 51 0.193 Pool from Sepharose 97 75 0.77

CL-4B column4 Heated enzyme from 97 65 0.67

step 35 Pool from DEAE- 25 57 2.3

Bio-Gel A6 Affi-Gel Blue column 6.2 35 5.7

pool AAffi-Gel Blue column 0.2 8.4 42

pool BAffi-Gel Blue column 0.2 8.3 41

pool C

1). There was a large amount of LPD-val which wasnot retained by the column (Fig. 1, pool A); pool Bwas eluted midway through the gradient, and pool Cwas eluted with 500 mM sodium chloride.pH optima. To determine the pH optima of the

lipoamide dehydrogenases in the direction of NADHoxidation, we used the components of the assay de-scribed previously (20), except that the buffers whichwe used were sodium acetate (pH 4.0 to 5.4), potassiumphosphate (pH 5.5 to 8.0), and Tris (pH 7.0 to 9.0) (allat a final concentration of 100mM). To determine thepH optima for NAD+ reduction, 50 mM Tris bufferwas used from pH 7.0 to 9.0 and 50 mM sodiumbicarbonate was used from pH 8.5 to 10.5.Gel electrophoresis. Sodium dodecyl sulfate-poly-

acrylamide gel electrophoresis was usually performedby the method of Laemmli and Favre (9), using 7.5%polyacrylamide gels. In some cases we used themethod of Weber et al. (26), in which a stacking gelwas not required. The molecular weights of the sub-units were determined by the method of Weber et al.(26) except that we used the buffer system of Laemmliand Favre (9). Lipoamide dehydrogenase activity wasdemonstrated in 5% polyacrylamide gels by using p-Nitro Blue Tetrazolium stain (22).

RESULTSPurification ofLPD-glu and LPD-val. The

objective of this study was to isolate lipoamidedehydrogenase from keto acid dehydrogenasecomplexes rather than free enzyme. P. putidagrown on glucose contains pyruvate and 2-keto-glutarate dehydrogenases but lacks branched-chain keto acid dehydrogenase (12). Whengrown on valine, P. putida contains all threeketo acid dehydrogenases, with two to threetimes as much branched-chain keto acid dehy-drogenase as the other two enzymes combined(23). Ultracentrifugation cleared the superna-

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642 SOKATCH ET AL.

tant fraction of lipoamide dehydrogenase in thepreparation from P. putida grown on valine, butmost of the lipoamide dehydrogenase remainedin the supernatant fraction in the preparationfrom cells grown on glucose, presumably in anuncomplexed form. Heating (Tables 1 and 2,step 4) released most of the lipoamide dehydro-genase from the complexes, which could then beisolated by ion-exchange chromatography andaffinity chromatography. The elution patternsfrom Affi-Gel Blue were different in the twopreparation procedures (Fig. 1). With the prep-aration made from cells grown on glucose, therewas one major peak at 150 ml. With the prepa-ration made from cells grown on valine, therewas a large amount of lipoamide dehydrogenasewhich was not retained by the column (Fig. 1,pool A). Pool B was eluted midway through thegradient at 60 ml, and pool C was eluted with500 mM sodium chloride. None of the purifiedlipoamide dehydrogenase preparations con-tained keto acid dehydrogenase activity. Thedata below show that the lipoamide dehydroge-nase of pool B was associated with growth onvaline and that pool C contained the same li-poamide dehydrogenase isolated from P. putidagrown on glucose.Electrophoresis of purified dihydroli-

poamide dehydrogenases. LPD-glu was a sin-gle protein which had a molecular weight of56,000, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Lipoamidedehydrogenase from pool B (Table 2) of thepreparation of cells grown on valine (LPD-val)was also a single protein but had a molecularweight of 49,000. These two proteins were sepa-rated easily by sodium dodecyl sulfate-poly-acrylamide gel electrophoresis (Fig. 2). Both pro-teins were responsible for their respective en-zyme activities since electrophoresis in 5% poly-acrylamide gels under nondenaturing conditionsshowed that the enzyme activity correspondedto the protein stain (Fig. 3).The proteins in the other two pools of lipoam-

ide dehydrogenase eluted from Affi-Gel Blue inthe preparation made from P. putida grown onvaline were also examined by sodium dodecylsulfate-polyacrylamide gel electrophoresis. PoolA (Table 2) contained six major and severalminor proteins and may have contained enzymewhich was still complexed. Rechromatographyof pool A on Affi-Gel Blue did not separate itinto pools A, B, and C. Pool C contained a singleprotein which had a molecular weight of 56,000and migrated with LPD-glu during sodium do-decyl sulfate-polyacrylamide gel electrophoresis.Therefore, pool C was probably LPD-glu.pH optima. There were distinct differences

in the pH optima of the two lipoamide dehydro-

a b c

FIG. 2. Sodiumdodecylsulfate-polyacrylamidegelelectrophoresis ofpurified LPD-val (lane a), LPD-glu(lane b), and a mixture of the two (lane c). Migrationwas from top to bottom.

genases. When pig heart lipoamide dehydroge-nase was assayed in the direction of NADHoxidation, NAD stimulated the reaction (15) andchanged the pH optimum from 6.2 to 5.6 (24).LPD-val behaved very much like the pig heartenzyme; the pH optimum decreased from 6.5 to5.7 when NAD+ was present. However, withLPD-glu the shift in the pH optimum in thepresence of NAD+ was only from 7.4 to 7.1. ThepH optima for LPD-glu and LPD-val for thereduction of NAD+ were 8.1 and 8.7, respec-tively, compared with 7.9 for the pig heart en-zyne (13).Kinetic constants for NADH oxidation.

Table 3 shows the kinetic constants for thelipoamide dehydrogenases for NADH oxidation.One difficulty of this study was that the solubil-ity of lipoamide in alcohol limited the range ofconcentrations which could be studied. There

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a b c d

FIG. 3. Protein and activity stains ofLPD-glu andLPD-val in 5% polyacrylamide gels. Lane a, LPD-glu, protein stain; lane b, LPD-glu, activity stain;lane c, LPD-val, protein stain; lane d, LPD-val, ac-tivity stain.

TABLE 3. Kinetic constants for lipoamidedehydrogenases assayed in the direction ofNADH

oxidation

Pres- V.Variable ence K. ( Hilsubstrate Enzyme of (mM) nun coeffi-

NAD per cientmg)

NADH LPD-glU + 0.035 39.9 0.94- 0.038 46.3 0.99

LPD-val + 0.015 78.8 0.93- 0.014 75.4 0.95

Lipoamide LPD-glu + 2.51 48.7 1.13- 2.59 42.7 1.23

LPD-val + 1.71 99 1.10- 2.78 118 0.98

were few differences between the two enzymeswith respect to the apparent Michaelis con-stants, maximum velocities, and-Hill coefficients.However, like the pig heart enzyme activity (15),there was a pronounced lag when LPD-glu ac-tivity was measured in the absence of NAD+.This lag was barely noticeable with LPD-val.Kinetic constants for NAD+ reduction.

The double-reciprocal plots for the reaction ofpig heart lipoamide dehydrogenase in the direc-tion of NAD+ reduction yielded a series of par-allel lines which fit a ping-pong mechanism ofcatalysis (14). Figure 4 shows double-reciprocalplots for NAD+ against fixed concentrations ofdihydrolipoamide for both LPD-glu and LPD-val at theirpH optima. The slopes and intercepts

-5 0 5 10 15 20

1

[NAD mM]

1

[NAD mM]

FIG. 4. Double-reciprocal plots for LPD-glu (A)and LPD-val (B) with the concentration of NADvaried against four fixed concentrations of dihydro-lipoamide. The concentrations of dihydrolipoamideused were 0.1 mM (A), 0.15 mM (5), 0.2 mM (0), and0.3mM (0).

were calculated by a computer program as de-scribed above and yielded a series of parallellines like those obtained with pig heart lipoam-ide dehydrogenase. The data from these lineswere used to calculate the bottom line (Fig. 4)at an infinite lipoamide concentration. Thesesame data were used to calculate a second familyof lines at an infinite NAD+ concentration withvarying concentrations of lipoamide. The Mi-chaelis constants, maximum velocities, and Hillcoefficients calculated in this study are shown inTable 4. The results were similar for the twoenzymes.

A. .05

>- .04

F-.03o 0

u .02

wi0- .01C')

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TABLE 4. Kinetic constants for lipoamidedehydrogenases assayed in the direction ofNAD+

reduction

vm HillVariable substrate Enzyme Km (umol/ coeffi-(mM) min per cient

mg) cen

NAD LPD-glu 0.37 286 0.99LPD-val 0.34 252 0.99

Dihydrolipoamide LPD-glu 0.77 238 0.99LPD-val 0.34 260 0.95

Cofactors. Both enzymes required NADHand lipoamide or NAD and dihydrolipoamidefor activity, depending on the direction of assay.Flavin adenine dinucleotide was identified as thebound cofactor of both lipoamide dehydrogen-ases by heating the enzymes at 100°C for 5 minand then chromatographing the freed cofactorby the method of Kilgour et al. (8).Requirement of branched-chain keto

acid dehydrogenase from P. putida forLPD-val. After we obtained a purified prepa-ration of branched-chain keto acid dehydroge-nase deficient in lipoamide dehydrogenase (23),it was possible to determine whether there was

a requirement for a specific lipoamide dehydro-genase. Table 5 shows that only LPD-val func-tioned with branched-chain keto acid dehydro-genase and that LPD-glu had no effect. It wasessential that this experiment be performed withfreshly prepared lipoamide dehydrogenases.Both LPD-glu and LPD-val were unstable, andaged preparations of LPD-val did not stimulatebranched-chain keto acid dehydrogenase as

much as fresh preparations, even when the samenumber of enzyme units was added.To determine the requirements of pyruvate

and 2-ketoglutarate dehydrogenases for lipoam-ide dehydrogenase, these enzymes were partiallypurified in an attempt to prepare complexesdeficient in lipoamide dehydrogenase. The pu-rification procedure started with 10 g of P. pu-tida grown on glucose. This procedure was sim-ilar to that used to prepare branched-chain ketoacid dehydrogenase and was carried to the stageof the DEAE-Sepharose CL-6B column (23).Both enzymes emerged from the DEAE-Seph-arose column at virtually the same position, so

that the enzyme pool contained both pyruvatedehydrogenase and 2-ketoglutarate dehydroge-nase. The specific activity of the partially puri-fied pyruvate dehydrogenase was 0.83 U/mg ofprotein when the preparation was assayed withpurified lipoamide dehydrogenase. Pyruvate de-hydrogenase was not deficient in lipoamide de-hydrogenase since it did not respond to eitherLPD-glu or LPD-val (Table 6). However, theactivity of 2-ketoglutarate dehydrogenase was

increased nearly fourfold by the addition ofLPD-glu but not LPD-val. It was clear that 2-ketoglutarate dehydrogenase has a specific re-quirement of LPD-glu. We drew no conclusionsconcerning the requirement for lipoamide de-hydrogenase by pyruvate dehydrogenase fromthe data in Table 6.

DISCUSSIONAs far as we know, this is the first evidence

that two functionally different lipoamide dehy-drogenases exist. LPD-val is produced only incells grown on valine as the principal carbon andenergy source and functions only with branched-chain keto acid dehydrogenase, an enzyme in-duced in P. putida during growth on branched-chain amino acids or their metabolites as thecarbon sources (7). LPD-glu is the sole lipoamidedehydrogenase produced during growth on glu-cose and does not function with branched-chainketo acid dehydrogenase. LPD-glu but not LPD-val was required for the activity of a partiallypurified preparation of 2-ketoglutarate dehydro-genase. The preparation of pyruvate dehydro-genase did not respond to either purified lipoam-ide dehydrogenase and was apparently not de-ficient in this component of the complex. How-ever, since P. putida grown on glucose containedonly LPD-glu, it seems likely that a requirementwill be demonstrated eventually.

TABLE 5. Stimulation of branched-chain keto aciddehydrogenase by LPD-vala

Activity(nmol ofSupplement to aay NADH

per min)

None ... ... ... .......... ... .. 1.1LPD-val ... ... ... .. ... 11.2LPD-glu ... ..... ... .. .... .. . 0.8

a Branched-chain keto acid dehydrogenase was pre-pared and assayed as described in the accompanyingpaper (23) with 0.15 U of LPD-glu or LPD-val.

TABLE 6. Stimulation of2-ketoglutaratedehydrogenase by LPD-glua

ActivityAssayconditions (nmol ofAssay conditions NADH

per min)

Pyruvate dehydrogenase ...8.1+LPD-glu ........... 9.3+LPD-val .. .......... 5.6

2-Ketoglutarate dehydrogenase 17.5+LPD-glu 66.2+LPD-val .... .......... .... 13.9a Each preparation of pyruvate dehydrogenase and

2-ketoglutarate dehydrogenase contained 3.65 ,ug ofprotein; 0.15 U amounts of lipoamide dehydrogenasewere added.

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It is clear that E. coli possesses a single lpdstructural gene (6) and a single lipoamide de-hydrogenase (16). Furthermore, since E. colidoes not grow in media containing branched-chain amino acids as the sole carbon source, thisorganism would not be expected to produceLPD-val. However, the situation with mamma-lian lipoamide dehydrogenase is less clear. Thereis considerable evidence for the existence of iso-zymes of pig heart lipoamide dehydrogenase(27), which have been separated by ion-exchangechromatography, electrophoresis, isoelectric fo-cusing, and affinity chromatography (11, 21, 25,28), but the catalytic properties, amino acid com-positions, and molecular weights of these iso-zymes have all been within the range of experi-mental error. The best explanation is that theseare conformational variations of the same pro-tein. There is evidence that some of these iso-zymes are associated with pyruvate dehydroge-nase and that others are associated with 2-ke-toglutarate dehydrogenase (21), but there is noreason to suspect that there is more than onelpd gene in mammals. Branched-chain keto aciddehydrogenase from bovine kidney lost lipoam-ide dehydrogenase activity during purification,but the activity of thisenzyme was restored byadding the usual lipoamide dehydrogenase;therefore, there is no evidence for the existenceof a separate lipoamide dehydrogenase forbranched-chain keto acid dehydrogenase inmammals (17).Because of the difference in molecular weight,

it is clear that LPD-glu and LPD-val are twodifferent proteins rather than conformationalisozymes. The specificity of LPD-val forbranched-chain keto acid dehydrogenase andthe fact that this enzyme is produced only whenbranched-chain keto acid dehydrogenase is in-auced are compelling evidence that LPD-val isproduced to serve branched-chain keto acid de-hydrogenase. Another important difference be-tween the two lipoamide dehydrogenases is thedifference in pH optima; however, in other re-spects, particularly the kinetic constants, thetwo enzymes are similar. LPD-val could be pro-duced by a proteolytic processing of LPD-glu, orit could be the result of a separate lpd gene.

ACKNOWLEDGMENTThis research was supported by Public Health Service

grant AM 21737 from the National Institutes of Health.

LITERATURE CITED1. Alwine, J. C., F. M. Russell, and K. N. Murray. 1973.

Characterization of an Escherichia coli mutant defi-cient in dihydrolipoyl dehydrogenase activity. J. Bac-teriol. 115:1-8.

2. Atkingson, D. E., J. A. Hathaway, and E. C. Smith.1965. Kinetics of regulatory enzymes. Kinetic order of

the yeast diphosphopyridine nucleotide isocitrate de-hydrogenase reaction and a model for the reaction. J.Biol. Chem. 240:2682-2690.

3. Barrera, C. R., G. Namihira, L. Hamilton, P. Munk,M. H. Eley, T. C. Linn, and L. J. Reed. 1972. a-Ketoacid dehydrogenase complexes. XVI. Studies on thesubunit structure of the pyruvate dehydrogenase com-plexes from bovine kidney and heart. Arch. Biochem.Biophys. 148:343-358.

4. Guest, J. R. 1974. Gene-protein relationships of the a-keto acid dehydrogenase complexes of Escherichia coliK12: chromosomal location of the lipoamide dehydro-genase gene. J. Gen. Microbiol. 80:523-532.

5. Guest, J. R. 1978. Aspects of the molecular biology oflipoamide dehydrogenase. Adv. Neurol. 21:219-244.

6. Guest, J. R., and I. T. Creaghan. 1973. Gene-proteinrelationships of the a-keto acid dehydrogenase com-plexes of Escherichia coli K12: isolation and character-ization of lipoamide dehydrogenase mutants. J. Gen.Microbiol. 75:197-210.

7. Jacobson, L. A., R. C. Bartholomaus, and L. C. Gun-salus. 1966. Repression of malic enzyme by acetate inPseudomonas. Biochem. Biophys. Res. Commun. 24:955-960.

8. Kilgour, G. L., S. P. Felton, and F. M. Huennekens.1957. Paper chromatography of flavins and flavin nu-cleotides. J. Am. Chem. Soc. 79:2254-2256.

9. Laemmli, U. K., and M. Favre. 1973. Maturation of thehead of bacteriophage T4. I. DNA packaging events. J.Mol. Biol. 80:575-599.

10. Lineweaver, J., and D. Burk. 1934. The determinationof enzyme dissociation constants. J. Am. Chem. Soc.56:658-666.

11. Lusty, C. J. 1963. Lipoyl dehydrogenase from beef livermitochondria. J. Biol. Chem. 238:3443-3452.

12. Marshall, V. P., and J. R. Sokatch. 1972. Regulation ofvaline catabolism in Pseudomonasputida. J. Bacteriol.110:1073-1081.

13. Massey, V. 1960. The identity of diaphorase and lipoyldehydrogenase. Biochim. Biophys. Acta 37:314-322.

14. Massey, V., Q. H. Gibson, and C. Veeger. 1960. Inter-mediates in the catalytic action of lipoyl dehydrogenase(diaphorase). Biochem. J. 77:341-351.

15. Massey, V., and C. Veeger. 1961. Studies on the reactionmechanism of lipoyl dehydrogenase. Biochim. Biophys.Acta 48:33-47.

16. Pettit, F. H., and L. J. Reed. 1967. a-Keto acid dehydro-genase complexes. VII. Comparison of dihydrolipoyldehydrogenases from pyruvate and a-ketoglutarate de-hydrogenase complexes of Escherichia coli. Proc. NatL.Acad. Sci. U.S.A. 58:1126-1130.

17. Pettit, F. H., S. J. Yeaman, and L. J. Reed. 1978.Purification and characterization of branched chain a-keto acid dehydrogenase complex of bovine kidney.Proc. Natl. Acad. Sci. U.S.A. 75:4881-4885.

18. Reed, L. J., and D. J. Cox. 1970. Multienzyme com-plexes, p. 213-240. In P. D. Boyer (ed.), The enzymes,vol. 1. Academic Press, Inc., New York.

19. Reed, L. J., M. Koike, M. E. Levitch, and F. R. Leach.1958. Studies on the nature and reactions of protein-bound lipoic acid. J. Biol. Chem. 23::143-158.

20. Reed, L J., and C. R. Wfllms. 1966. Purification andresolution of the pyruvate dehydrogenase complex(Escherichia coli). Methods Enzymol. 9:247-265.

21. Sakurai, Y., Y. Fukuyoshi, M. Hamada, T. Hayak-awa, and M. Koike. 1970. Mammalian a-keto aciddehydrogenase complexes. VI. Nature of the multipleforms of pig heart lipoamide dehydrogenase. J. Biol.Chem. 245:4453-4462.

22. Scouten, W. H., and L. R. McManus. 1971. Microbiallipoamide dehydrogenase. Purification and some char-acteristics of the enzyme derived from selected micro-organisms. Biochim. Biophys. Acta 227:248-263.

VOL. 148, 1981

on August 26, 2020 by guest

http://jb.asm.org/

Dow

nloaded from

646 SOKATCH ET AL.

23. Sokatch, J. R., V. McCully, and C. M. Roberts. 1981.Purification of a branched-chain keto acid dehydroge-nase from Pseudomonas putida. J. Bacteriol. 148:647-652.

24. van Muiswinkel-Voetberg, H., and C. Veeger. 1973.Conformational studies on lipoamide dehydrogenasefrom pig heart. IV. The binding ofNAD+ to non-equiv-alent sites. Eur. J. Biochem. 33:285-291.

25. Visser, J., and M. Strating. 1975. Separation of lipoam-ide dehydrogenase isoenzymes by affinity chromatog-

J. BACTERIOL.

raphy. Biochim. Biophys. Acta 384:69-80.26. Weber, K., J. R. Pringle, and M. Osborn. 1972. Mea-

surement of molecular weight by electrophoresis inSDS-acrylamide gel. Methods Enzymol. 26:3-27.

27. Williams, C. H. 1976. Flavin-containing dehydrogenases,p. 89-173. In P. D. Boyer (ed.), The enzymes, vol. 13.Academic Press, Inc., New York.

28. Wilson, J. E. 1971. A comparative study of the multipleforms of pig heart lipoyl dehydrogenase. Arch. Biochem.Biophys. 144:216-223.

on August 26, 2020 by guest

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Dow

nloaded from