JOURNAL OF BACTROLOGY, June 1972, p. 1073-1081Copyright 0 1972 American Society for Microbiology

Vol. 110, No. 3Printed in U.S.A.

Regulation of Valine Catabolism inPseudomonas putida

VINCENT DEPAUL MARSHALL AND JOHN R. SOKATCH

Department of Microbiology and Immunology, University of Oklahoma Health Sciences Center, OklahomaCity, Oklahoma 73104

Received for publication 27 March 1972

The activities of six enzymes which take part in the oxidation of valine byPseudomonas putida were measured under various conditions of growth. Theformation of four of the six enzymes was induced by growth on D- or L-valine:D-amino acid dehydrogenase, branched-chain keto acid dehydrogenase, 3-hy-droxyisobutyrate dehydrogenase, and methylmalonate semialdehyde dehydro-genase. Branched-chain amino acid transaminase and isobutyryl-CoA dehydro-genase were synthesized constitutively. D-Amino acid dehydrogenase andbranched-chain keto acid dehydrogenase were induced during growth on valine,leucine, and isoleucine, and these enzymes were assumed to be common to themetabolism of all three branched-chain amino acids. The segment of thepathway required for oxidation of isobutyrate was induced by growth on isobu-tyrate or 3-hydroxyisobutyrate without formation of the preceding enzymes. D-

Amino acid dehydrogenase was induced by growth on L-alanine without forma-tion of other enzymes required for the catabolism of valine. D-Valine was a

more effective inducer of D-amino acid dehydrogenase than was L-valine.Therefore, the valine catabolic pathway was induced in three separate seg-

ments: (i) 1-amino acid dehydrogenase, (ii) branched-chain keto acid dehydro-genase, and (iii) 3-hydroxyisobutyrate dehydrogenase plus methylmalonatesemialdehyde dehydrogenase. In a study of the kinetics of formation of theinducible enzymes, it was found that 3-hydroxyisobutyrate and methylmal-onate semialdehyde dehydrogenases were coordinately induced. Induction ofenzymes of the valine catabolic pathway was studied in a mutant that had lostthe ability to grow on all three branched-chain amino acids. Strain PpM2106had lowered levels of branched-chain amino acid transaminase and completelylacked branched-chain keto acid dehydrogenase when grown in medium whichcontained valine. Addition of 2-ketoisovalerate, 2-ketoisocaproate, or 2-keto-3-methylvalerate to the growth medium of strain PpM2106 resulted in inductionof normal levels of branched-chain keto acid dehydrogenase; therefore, thebranched-chain keto acids were the actual inducers of branched-chain keto aciddehydrogenase.

Regulation of catabolic pathways for aminoacids in bacteria must provide a mechanismfor enzyme induction which is able to ignoreamino acids of the amino acid pool, but whichcan respond to the amino acids in the me-dium. Histidine and tryptophan are notusually inducers of enzymes for their catabo-lism; rather the inducer is an early metaboliteformed from these amino acids. Schlesinger,Scotto, and Magasanik (19) found that uro-canate and not histidine was the inducer forthe formation of enzymes of histidine catabo-lism in Aerobacter aerogenes. This conclusionwas based on the finding that mutants which

lacked histidine ammonia-lyase formed theother enzymes of the histidine catatolicpathway with urocanate in the medium, butnot with histidine. Newell and Lessie (9) re-cently reported that urocanate was the inducerof histidine-degrading enzymes in Pseudo-monas aeruginosa as well. Brill and Magasanik(2) determined that histidine ammonia-lyaseand urocanase were coordinately induced inSalmonella typhimurium and most probablyby urocanate rather than histidine.

In contrast to the relatively simple situationwith histidine, several inductive events arerequired for induction of the complete trypto-

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phan catabolic pathway. The first two enzymesof the pathway, tryptophan pyrrolase and kyn-urenine formylase, were induced coordinatelyin Pseudomonas fluorescens by kynurenine(15). Kynureninase, the third enzyme, was alsoinduced by kynurenine, but at a rate differentfrom that of the first two enzymes, and wasthe first sequentially induced enzyme. There-fore, there were two inductive events in thesynthesis of the first three enzymes of trypto-phan oxidation. Tryptophan itself was not aninducer of the pathway; Palleroni and Stanier(15) were able to prove this with a mutantlacking tryptophan pyrrolase. Kynurenine in-duced the formation of kynurenine formylaseand kynureninase in the mutant, but trypto-phan did not. Therefore, kynurenine appearsto be the inducer of this segment of thepathway.Many species of Pseudomonas oxidize tryp-

tophan by way of anthranilate and catechol.Regulation of catechol and p-hydroxybenzoatemetabolism was studied in Pseudomonas pu-tida by Ornston (13) and in P. aeruginosa byKemp and Hegeman (6), and the same regula-tory mechanisms appear to occur in both orga-nisms. There are at least four inductive eventsbetween anthranilate and f3-ketoadipate.Ornston has recently published a thorough re-view on regulation of catabolic pathways whichcovers this subject in detail (14).There are two known instances where an

amino acid is the inducer of its own catabolicpathway. Mutants of Bacillus subtilis thatlacked histidine ammonia-lyase produced uro-canase and formiminoglutamate hydrolasewhen grown with histidine (3). A mutant of S.typhimurium that lacked proline oxidase, thefirst enzyme of the proline catabolic pathway,formed pyrroline-5-carboxylate dehydrogenasewhen grown with proline (4).The object of the present study was to inves-

tigate the regulation of valine catabolism in P.putida. Previous studies on valine catabolismfrom our laboratory were made with P. aerugi-nosa, and a proposal for the pathway of valineoxidation in this species is shown in Fig. 1 (1).The present studies were made with P. putida,because we had access to mutants of this orga-nism blocked in the metabolism of valine (R.R. Martin, M.S. thesis, Univ. of OklahomaHealth Sciences Center, Oklahoma City, 1969).Data presented in this paper establish that thecatabolic pathway for valine in P. putida canbe induced in three separate segments: (i) D-amino acid dehydrogenase, (ii) branched-chainketo acid dehydrogenase, and (iii) 3-hydroxyi-sobutyrate dehydrogenase and methylmalonate

semialdehyde dehydrogenase. Inducers forbranched-chain keto acid dehydrogenase, thefirst enzyme unique to the metabolism of thebranched-chain amino acids, were the ketoacids derived from the branched-chain aminoacids. Valine was not an inducer of this en-zyme.

MATERIALS AND METHODSOrganisms. P. putida, strain PpG2, was obtained

from I. C. Gunsalus at the University of Illinois.Mutants of P. putida unable to grow on valine as thesole source of carbon were obtained from R. R.Martin.

Cultural conditions. P. putida was grown at 30 Cin the basal medium of Jacobson (L. A. Jacobson,Ph.D. thesis, Univ. of Illinois, Urbana, 1967) with thecarbon sources added at the appropriate concentra-tion. It was necessary to supplement media con-taining valine with 0.005% L-isoleucine to overcometoxicity of valine to P. putida (L. A. Jacobson, Ph.D.thesis). P. aeruginosa was grown in the same me-dium at 37 C.

Stock cultures of P. putida or P. aeruginosa wereused to inoculate 5-ml amounts of complete mediumwhich were incubated overnight at 30 and 37 C, re-spectively, with shaking. The 5-ml cultures wereadded to 1-liter batches of complete medium, whichwere incubated with aeration to the end of the logphase at 30 or 37 C. The cell crop was harvested bycentrifugation and used as a source of enzyme formost of the induction experiments. Larger amountsof culture were needed to study the kinetics of en-zyme induction and were obtained by inoculating 1liter of culture into 14 liters of medium in a fer-mentor (Fermentation Design, Inc.) with aeration at30 or 37 C to the end of log phase of growth. Growthwas followed by diluting samples of the culture five-fold with water and reading at 660 nm with a Bausch& Lomb Spectronic 20 spectrophotometer.

Synthesis of substrates. 3-Hydroxyisobutyratewas synthesized by the method of Robinson andCoon (17), and methylmalonate semialdehyde wasprepared by the technique of Kupiecki and Coon (7).Isobutyryl-coenzyme A (CoA), methacrylyl-CoA, and3-hydroxyisobutyryl-CoA were synthesized bymethods described by Stadtman (22) which werebased on the method of Simon and Shemin (20). Allother reagents were commercial preparations.Enzyme preparation. For the preparation of the

enzyme extracts, 1 to 5 g of cell paste was suspendedin 10 ml of 0.05 M potassium phosphate buffer, pH7.5, subjected to sonic disruption with a Bransonmodel S75 sonifier for 5 to 10 min and centrifugedfor 15 min at 10,000 x g, and the supernatant fluidused as the source of enzyme. Protein concentrationswere determined by the method of Warburg andChristian (24).Assays of enzymatic activity. All specific activi-

ties are expressed as nanomoles of product formedper minute per milligram of protein.D-Amino acid dehydrogenase was assayed by the

method of Norton, Bulmer, and Sokatch (10) using a

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CH3 IX H2CH - CH - COOH

CH13 - \ Transaminationl.a

L-Valine CH3CH - CO - COOH 1

2H CH3CH3 , CH - CH - COOH I.b 2- Ketoisovalerate

1-1CH3 NH2

D-Valine

CH3,,C - CO - CoA

CH2

Methacrylyl - CoA

-2H, CoA CH3 \

CHCH - CO - l:oA + C02

CH3IyIsobutyryl - CoA

CH3\C

CH - COOHCHO

NAD, CoA

VIl

CH3CH2 + C02CO - CoA

ATP, C02 CH3_ CH - COOH

Vill CO - CoA

Methylmalonate Propionyl - CoA Methylmalonyl - CoA

semialdehydeFIG. 1. Pathway for the metabolism of D- and L-valine in pseudomonads. The enzymatic reactions and

enzymes whose activities were measured in this study were: (I. a) branched-chain amino acid transaminase;(I. b) D-amino acid dehydrogenase; (II) branched-chain keto acid dehydrogenase; (III) isobutyrl-CoA dehydro-genase; (VI) 3-hydroxyisobutyrate dehydrogenase; and (VII) methylmalonate semialdehyde dehydrogenase.

spectrophotometer (Beckman model DU) equippedwith a multiple absorbance recorder (Gilford model2000). The spectrophotometer was equipped withthermospacers through which water circulated at a

temperature of 37 C.Branched-chain amino acid transaminase (EC

2.6.1.6) was measured by the assay of Taylor andJenkins (23).The assay for branched-chain keto acid dehydro-

genase was developed during the course of thepresent study. The oxidation of 2-ketoisovalerateand other branched-chain keto acids was followed bymeasuring the production of reduced nicotinamideadenine dinucleotide (NADH) spectrophotomet-rically at 340 nm. The standard assay was per-

formed with two cuvettes; the first cuvette con-

tained 10 gmoles of 2-ketoisovalerate, enzyme, 100gmoles of potassium phosphate buffer (pH 8.0), 10gmoles of NAD, 1 umole of CoA, 10 Amoles of dithi-othreitol, and enough water to bring the total volumeto 1 ml. The second cuvette contained all reagents

except substrate, and was used to adjust the spectro-photometer to zero absorbance. The reaction was

started with 0.1 to 0.65 mg of enzyme protein. Oneunit of enzyme activity was defined as 1 nmole ofNADH produced per min.

Isobutyryl-CoA dehydrogenase was assayed by themethod of Robinson et al. (18). The assay of Rob-inson and Coon (13) was used to measure the ac-

tivity of 3-hydroxyisobutyrate dehydrogenase (EC1.1.1.31). The assay for activity of methylmalonatesemialdehyde dehydrogenase was the assay devel-oped by Sokatch, Sanders, and Marshall (21).

RESULTS

Assay of branched-chain keto acid dehy-drogenase. The assay that was developed forbranched-chain keto acid dehydrogenase was

based on measurement of the change in ab-sorption at 340 nm due to the reduction ofNAD. 2-Ketoisovalerate, coenzyme A, and a

-2H

III

H20 CH3 X H20 CH3V OVCH-HCO-CoA CH-COOH+CoA

IV CH20H1-

V CH20H

3 - Hydroxyisobutyryl - CoA 3 - Hydroxyisobutyrate

NAD

VI

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reducing agent such as mercaptoethanol or

dithiothreitol were necessary for enzyme ac-

tivity. The crude extract also reduced NADwith 2-ketoisovalerate, 2-keto-3-methylvaler-ate, 2-ketoisocaproate, 2-ketoglutarate, andpyruvate under these conditions. The productof the reaction with 2-ketoisovalerate was

identified as isobutyryl-CoA. This identifica-tion was accomplished by separation of thecoenzyme A ester from the reaction mixture bythe procedure of Yamada and Jacoby (25) andconversion of the coenzyme A ester to the hy-droxamate derivative. Isobutyrohydroxamatewas the only hydroxamic acid detected in thereaction mixture. The activity of branched-chain keto acid dehydrogenase was directlyproportional to the amount of protein in theassay to approximately 20 nmoles NADHformed per minute.

Inducible enzymes of the valine catabolicpathway. Jacobson (Ph.D. thesis, Univ. of Illi-nois, Urbana, 1967) found that growth of P.putida on L-valine was stimulated by a trace ofisoleucine in the medium. We have confirmedthis observation and routinely included 0.005%isoleucine in media containing valine. P. pu-

tida will also grow with D-valine as the carbonand energy source if isoleucine is present inthe medium, but the generation time is abouttwice as long as with L-valine. P. aeruginosaalso grows more rapidly with L-valine, but iso-leucine has no effect on the growth rate.

It is clear from the data shown in Tables 1and 2 that the growth of P. putida and P. aeru-

ginosa on valine resulted in the induction of D-amino acid dehydrogenase, branched-chainketo acid dehydrogenase, 3-hydroxyisobutyratedehydrogenase, and methylmalonate semialde-hyde dehydrogenase. On the other hand,branched-chain amino acid transaminase andisobutyryl-CoA dehydrogenase were constitu-tive enzymes in both P. putida and P. aerugi-

nosa. P. putida contained measurable levels of3-hydroxyisobutyrate dehydrogenase andmethylmalonate semialdehyde dehydrogenasewhen grown on glucose, succinate, or L-gluta-mate, but these two enzymes were not de-tected in P. aeruginosa grown under theseconditions. The level of branched-chain ketoacid dehydrogenase in extracts made from P.putida grown on valine was also markedlyhigher than those from P. aeruginosa.Common pathway. There is evidence for a

common pathway in the early reactions in themetabolism of all three branched-chain aminoacids. It can be seen from the data in Table 3that growth of P. putida on any one of thethree branched-chain amino acids resulted inthe induction of D-amino acid dehydrogenaseand branched-chain keto acid dehydrogenase.However, the levels of 3-hydroxyisobutyratedehydrogenase and methylmalonate semialde-hyde dehydrogenase were low in those orga-nisms grown on isoleucine and leucine. There-fore, the segment of the pathway up to thebranched-chain keto acid dehydrogenase canbe induced separately from the remainder ofthe pathway.Induction of the isobutyrate segment of

pathway. When P. putida was grown withisobutyrate as the carbon source, 3-hydroxyi-sobutyrate dehydrogenase and methylmalonatesemialdehyde dehydrogenase were the onlytwo enzymes of the valine catabolic pathwaythat could be detected in amounts distinctlyhigher than those observed in cells grown on

glucose (Table 4). The conclusion made fromthese data is that these two enzymes at leastcan be induced separately from enzymes in theearlier part of the pathway. Growth of P. pu-tida on propionate resulted in slightly higherlevels of dehydrogenases for 3-hydroxyiso-butyrate and methylmalonate semialdehyde.Growth of P. putida with 3-hydroxyisobutyrate

TABLE 1. Comparison of activity of valine catabolic enzymes in Pseudomonas putida strain PpG2 andPseudomonas aeruginosa

Specific activity when carbon source for growth was:

Enzyme 0.3% L-Valine + 0.005% 0.3% D-GlucoseL-isoleucine

P. putida P. aeruginosa P. putida P. aeruginosa

D-Amino acid dehydrogenase 2.5 2.5 0 0Branched-chain amino acid transaminase 30 30 33 29Branched-chain keto acid dehydrogenase 40 7 0 . 0Isobutyryl-CoA dehydrogenase 1.5 1.5 1.7 1.73-Hydroxyisobutyrate dehydrogenase 200 100 20 0Methylmalonate semialdehyde dehydrogenase 100 62 10 0

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TABLE 2. Levels of valine catabolic enzymes in Pseudomonas putida strain PpG2 grown on valine,glutamate, succinate, and glucose

Specific activity when carbon source for growth was:

Enzyme 0.3% L-Valine 0.3% L-+ 0.005% Glutamate 0.3% Succinate 0.3% Glucose

L-isoleucine

D-Amino acid dehydrogenase 4 0 0 0Branched-chain amino acid transaminase 27 27 27 28Branched-chain keto acid dehydrogenase 29 0 0 0Isobutyryl-CoA dehydrogenase 1 1 1 13-Hydroxyisobutyrate dehydrogenase 170 20 10 10Methylmalonate semialdehyde dehydrogenase 48 12 7 7

TABLE 3. Induction of valine catabolic enzymes in Pseudomonas putida strain PpG2 by branched-chainamino acids

Specific activity when carbon source for growth was:

Enzyme 0.3% L-Valine 0.3% L-+ 0.005% Isoleucine 0.3% L-Leucine 0.3% Glucose

L-Isoleucine

D-Amino acid dehydrogenase 1 1 1 1Branched-chain keto acid dehydrogenase 80 200 40 03-Hydroxyisobutyrate dehydrogenase 160 66 22 15Methylmalonate semialdehyde dehydrogenase 50 27 12 8

TABLE 4. Induction of D-amino acid dehydrogenase, 3-hydroxyisobutyrate dehydrogenase andmethylmalonate semialdehyde dehydrogenase

Specific activity when carbon source for growth was:

Enzyme 0.3% L- 0.3% 3-Valine + 0.3% Hydroxy 0.3% Pro- 0.3% 0.3%0.005% Isobutyrate isobutyrate pionate L-Alanine Glucose

L-isoleucine

D-Amino acid dehydrogenase 1 0 0 0 1.4 0Branched-chain keto acid dehydrogenase 50 0 12 0 3 03-Hydroxyisobutyrate dehydrogenase 200 330 540 60 64 23Methylmalonate semialdehyde dehydro- 70 140 72 50 9 17

genase

also resulted in elevated levels of 3-hydroxyi-sobutyrate dehydrogenase and methylmalonatesemialdehyde dehydrogenase.Induction of D-amino acid dehydrogenase.

D-Amino acid dehydrogenase of P. aeruginosahas a broad specificity similar to that of thekidney enzyme (8). It seemed reasonable thatthis enzyme was not unique to valine metabo-lism and therefore it should be possible to in-duce its formation separately from the otherenzymes of the valine catabolic pathway. Thedata in Table 4 show that this was indeed thecase, since P. putida grown with L-alanineformed D-amino acid dehydrogenase at levelsusually observed during growth on valine,whereas the activities of the other inducibleenzymes of the pathway were low. The specific

activity of D-amino acid dehydrogenase ob-tained when the organism was grown with D-valine was considerably higher than with L-va-line (Table 5); this was unexpected since bothisomers of valine stimulated the formation ofthe same level of this enzyme in P. aeruginosa(8). Extracts of P. putida were examined forthe presence of valine racemase by the samemethod used previously (11), and the resultwas negative as it was in the case of P. aerugi-nosa. L-Glutamate was included in the me-dium in order to obtain the same growth rateswith D- and L-valine.Kinetics of enzyme induction. The kinetics

of induction of the four inducible enzymes ofthe valine catabolic pathway were studied todetermine if there was coordinate induction of

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any of the enzymes. P. putida was grown in 14liters of medium with L-glutamate as thecarbon source. Near the middle of the loga-rithmic phase of growth, usually about 3.25 hrafter inoculation, DL-valine was added to themedium to yield a final concentration of 0.1%and DL-isoleucine to yield a concentration of0.005%. Samples were taken from the mediumat intervals thereafter and frozen quickly withdry ice and acetone. The samples were thawed,the cells were disrupted by sonic oscillationand centrifuged, and activities of the four in-ducible enzymes were determined.The data were expressed as per cent of the

highest specific activity of the enzyme ob-tained in the experiment, a method used sev-

eral years ago by Jacob and Monod (5). It was

then possible to compare rates of induction ofenzymes with widely different specific activi-ties. Calculation of slope, correlation coeffi-cient and Y intercept were performed on a

IBM model 1800 computer, programmed byDonald Parker of the Department of Biostatis-tics and Epidemiology, University of Okla-homa Health Sciences Center. We have as-

sumed coordinate induction when the slope ofsuch a curve was 1.0 and the Y intercept was

zero. By these criteria, coordinate inductionwas obtained only in the case of 3-hydroxyiso-butyrate and methylmalonate semialdehydedehydrogenases. All other combinations pro-duced plots that did not go through zero or

had slopes significantly different from 1.0(Table 6).Enzyme induction in mutants. Strain

PpM2106 has lost the ability to grow on valine,isoleucine, and leucine, has a slightly impairedability to grow on 2-ketoisovalerate, and growsnormally on isobutyrate and 3-hydroxyiso-butyrate. The enzymatic lesion in this orga-nism appears to be in the formation ofbranched-chain amino acid transaminase(Table 7). The specific activities for the trans-aminase of the mutant were 40 to 60% of the

level found in the wild type but were never

undetectable. When strain PpM2106 was

grown in the presence of valine and L-gluta-mate as the carbon source, there was no de-tectable branched-chain keto acid dehydro-genase (Table 7). It is interesting, however,that nearly normal levels of 3-hydroxyiso-butyrate and methylmalonate semialdehydedehydrogenases were found. Addition of 2-ke-toisovalerate, 2-ketoisocaproate, or 2-keto-3-methylvalerate to the medium caused the in-duction of branched-chain keto acid dehydro-genase (Table 8). These data provide evidencethat the inducers of branched-chain keto aciddehydrogenase are the keto acids derived fromvaline, leucine, and isoleucine. Valine isclearly not an inducer (Table 7), and since iso-butyrate and 3-hydroxyisobutyrate were notinducers (Table 4), the branched-chain ketoacids themselves must be the inducers of thisenzyme.

DISCUSSIONThe evidence presented in this paper dem-

onstrates that there are at least three separateinductive events in formation of enzymes forthe catabolism of D-valine and two for L-va-line: one for the D-amino acid dehydrogenase,one for branched-chain keto acid dehydrogen-ase, and one for 3-hydroxyisobutyrate andmethylmalonate semialdehyde dehydrogen-ases.The observed pattern of enzyme induction

appears to meet the needs of the cell with re-

spect to the metabolism of valine. Branched-chain amino acid transaminase is required forsynthesis of all three branched-chain aminoacids as well as for their catabolism; therefore,the constitutive character of this enzyme isappropriate. D-Amino acid dehydrogenase isrequired only when relatively rare D-branched-chain amino acids are present in the medium,and therefore its inducible nature is consistentwith this need. This enzyme may also be nec-

TABLE 5. Induction of valine catabolic enzymes by L- and D-valine in Pseudomonas putida strain PpG2

Specific activity after growth on:

Enzyme 0.1% L-Valine, 0.1% D-Valine,0.005% L-iSO- 0.005% L-iSo- 0.3%leucine, 0.3% leucine, 0.3% L-GlutamateL-glutamate L-glutamate

D-Amino acid dehydrogenase 1.8 12 0Branched-chain amino acid transaminase 96 81 70Branched-chain keto acid dehydrogenase 60 16 93-Hydroxyisobutyrate dehydrogenase 125 133 60Methylmalonate semialdehyde dehydrogenase 92 100 34

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TABLE 6. Kinetics of induction of enzymes in the valine catabolic pathway

Corre-X axis Y axis Slope lation Y

coeffi- interceptcient

3-Hydroxyisobutyrate dehydrogenase D-Amino acid dehydrogenase 1.10 0.87 21.8D-Amino acid dehydrogenase Branched-chain keto acid dehydro- 0.68 0.91 0.54

genase3-Hydroxyisobutyrate dehydrogenase Methylmalonate semialdehyde dehy- 1.06 0.98 0.47

drogenase3-Hydroxyisobutyrate dehydrogenase Branched-chain keto acid dehydro- 0.91 0.96 11.22

genaseMethylmalonate semialdehyde dehy- D-Amino acid dehydrogenase 1.03 0.88 21.9drogenase

Methylmalonate semialdehyde dehy- Branched-chain keto acid dehydro- 0.83 0.94 12.1drogenase genase

TABLE 7. Induction of valine catabolic enzymes in Pseudomonas putida strain PpM2106

Specific activity

P. putida PpM2106 grown on: P. ongrown on:

Enzyme 0.1% L-Valine, 0 1% 2-Keto 0.1% L-Valine,0.005% L- 012Keo0.005% L-isoleucine, isovalerate 0.3% isoleucine,and 0.3% an 0.3% L-Glutamate and 0.3%

L-glutamate L-glutamate L-glUtamate

D-Amino acid dehydrogenase 3 0 0 3Branched-chain amino acid transaminase 19 35 22 51Branched-chain keto acid dehydrogenase 0 21 0 25Isobutyryl-CoA dehydrogenase 1 1 1 13-Hydroxyisobutyrate dehydrogenase 120 50 20 160Methylmalonate semialdehyde dehydrogenase 26 12 7 27

essary for the metabolism of other D-aminoacids, such as D-alanine and D-phenylalanine.Branched-chain keto acid dehydrogenase isnecessary only during the catabolism ofbranched-chain amino acids, and the enzymeis formed only under these conditions. It isinteresting that the inducers of this enzymeare the keto acid substrates for the enzymeand not the branched-chain amino acids them-selves. This relationship is analogous to sys-tems for induction of enzymes of tryptophanand histidine catabolism in Pseudomonas,where early intermediates formed from theamino acids are the inducers and not theamino acids. Finally, 3-hydroxyisobutyratedehydrogenase and methylmalonate semialde-hyde dehydrogenase are both necessary for,and unique to, metabolism of isobutyrate, andthese two enzymes are coordinately induced.Presumably, the genes that code for the syn-thesis of these two enzymes are located closetogether. While isobutyryl-CoA is a product ofvaline metabolism, it is also an intermediate

in the metabolism of at least one other com-pound, camphor (Jacobson, Ph.D. thesis, Univ.of Illinois, Urbana, 1967), and this segment ofthe valine pathway can be induced separatelyfrom the earlier part of the pathway.We believe that the early enzymes of the

pathway, D-amino acid dehydrogenase,branched-chain amino acid transaminase, andbranched-chain keto acid dehydrogenase, areenzymes of a pathway common to metabolismof all three branched-chain amino acids. D-Amino acid dehydrogenase (8) and branched-chain amino acid transaminase (12) are ca-pable of deaminating the D and L isomers of allthree branched-chain amino acids. Unfraction-ated extracts of wild-type P. putida are ca-pable of oxidizing all three keto acids derivedfrom the branched-chain amino acids whengrown on valine, an observation that is con-sistent with the concept of a commonpathway. Evidence will be presented in a sub-sequent publication which will show that amutation causes loss of ability to oxidize all

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TABLE 8. Induction of branched-chain keto aciddehydrogenase in Pseudomonas putida strain

PpM2106

Specificactivity of

Carbon source for growth branched-chain ketoacid dehy-drogenase

0.1% L-Valine + 0.005% L-isoleucine + 00.3% L-glutamate

0.1% 2-Ketoisovalerate + 0.3% L-gluta- 15mate

0.1% 2-Ketoisocaproate + 0.3% L-gluta- 10mate

0.1% 2-Keto-3-methylvalerate + 0.3% L- 15glutamate

0.3% L-Glutamate 0

three branched-chain keto acids (R. R. Martin,V. P. Marshall, J. R. Sokatch, and L. Unger,manuscript in preparation). Furthermore, thismutation, as well as the mutation that appearsto affect the transaminase of strain PpM2106,causes a simultaneous loss of ability to grow onall three branched-chain amino acids.The difference between the two species of

Pseudomonas in their response to the presenceof D-valine in the medium is interesting. P.putida produces much higher levels of D-aminoacid dehydrogenase with D-valine than with L-valine, whereas there is no difference in en-

zyme levels in the case of P. aeruginosa underthese conditions. Since neither organism has a

detectable racemase for valine, the differencein response may be due to inducer recognitionsites; the recognition site in P. aeruginosa re-

sponds to both D- and L-amino acids, whilethat in P. putida recognizes the D-amino acidmuch more efficiently. This problem was in-vestigated further by another student in our

laboratory who found that all the D-aminoacids which served as substrates for D-aminoacid dehydrogenase were much better inducersof the enzyme in P. putida than were the cor-

responding L-amino acids (I. A-L. Yeh, M.S.thesis, Univ. of Oklahoma Health SciencesCenter, Oklahoma City, 1971). P. aeruginosaresponded with the synthesis of comparablelevels to either isomer of these same aminoacids.

ACKNOWLEDGMENTS

This research was supported by Public Health Servicegrant no. 5 RO1 AM 09750 from the National Institute ofArthritis and Metabolic Diseases, National Science Founda-tion grant no. GB 23346, Public Health Service Career De-velopment Award no. 5 K03 GM 18343 from the NationalInstitute of General Medical Sciences to John R. Sokatch,

and Predoctoral Fellowship Award no. 1 FO1 GM 41376 toVincent deP. Marshall.

LITERATURE CITED

1. Bannerjee, D., L. E. Sanders, and J. R. Sokatch. 1970.Properties of purified methylmalonate semialdehydedehydrogenase of Pseudomonas aeruginosa. J. Biol.Chem. 245:1828-1835.

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3. Chasin, L. A., and B. Magasanik. 1968. Induction andrepression of the histidine-degrading enzymes of Ba-cillus subtilis. J. Biol. Chem. 243:5165-5178.

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