branched-chain amino acid catabolism bacteria · (branched-chain amino acid 2-ketoglutarate...

BACTERIOLOGICAL REVIEWS, Mar. 1976, p. 42-54Copyright C 1976 American Society for Microbiology

Vol. 40, No. 1Printed in U.S.A.

Branched-Chain Amino Acid Catabolism in BacteriaLINDA K. MASSEY,* JOHN R. SOKATCH, AND ROBERT S. CONRAD

Division of Medical Biology, Oklahoma College of Osteopathic Medicine and Surgery, Tulsa, Oklahoma74119,* and Department of Microbiology and Immunology, University ofOklahoma Health Sciences Center,

Oklahoma City, Oklahoma 73190

INTRODUCTION ............................................................. 42

ENZYMES COMMON TO CATABOLISM OF ALL THREE BRANCHED-CHAIN AMINO ACIDS ....................................................43

Branched-Chain Amino Acid Transaminase ................................... 44

L-Amino Acid Oxidase ....................................................... 44

D-Amino Acid Dehydrogenase ................................................ 45

Branched-Chain Keto Acid Dehydrogenase ................................... 45

Branched-Chain Acyl-CoA Dehydrogenase .................................... 46

ENZYMES SPECIFIC FOR INDIVIDUAL AMINO ACID PATHWAYS ........ 46

Leucine .................................................................... 46

Isoleucine .................................................................. 47

Valine ..................................................................... 47

SPECIFICITY OF ENOYL-CoA HYDRATASES ............................... 48

CATABOLIC PATHWAYS CONVERGING WITH BRANCHED-CHAIN AMINO ACID PATHWAYS ....................................... 48

Geraniol and Farnesol ....................................................... 48

Panthothenate .............................................................. 49

Mevalonate and 3-Hydroxy-3-Methylglutarate ................................. 49

Camphor ................................................................ 50

INHIBITION OF GROWTH BY BRANCHED-CHAIN AMINO ACIDS ......... 50

REGULATION OF DIVERGENT CATABOLIC PATHWAYS ................... 51

LITERATURE CITED ........................................................ 51

INTRODUCTION

The enzymatic conversions necessary for thecatabolism of branched-chain amino acids havebeen reported to occur in a wide variety ofbacteria. However, most studies have beendone using several species of Pseudomonas.Pseudomonads are richly endowed with ex-

traordinary nutritional versatility, which ena-

bles them to catabolize a diverse array of or-

ganic compounds (64), including the branched-chain amino acids. In a recent review of theregulation of catabolic pathways in Pseudomo-nas (44), Ornston discussed the history, meth-ods of approach, and evolutionary forces affect-ing catabolic pathways. This review will sum-

marize the bacterial catabolism of leucine, iso-leucine, valine, and several compounds whosecatabolic pathways converge with those of thebranched-chain amino acids. Readers inter-ested in mammalian catabolism of amino acidcarbon skeletons are referred to a review byRodwell (54). The data published in recentyears on branched-chain amino acid catabolismleave little doubt as to the authenticity of theproposed metabolic scheme (Fig. 1).The implication of defects in branched-chain

amino acid catabolism as foci for inborn errors

of metabolism has given both clinical relevance

and research impetus to the elucidation of thesepathways. The initial clinical observation ofthis phenomenon was made by pediatricians(43, 63), who correlated the uremic excretion ofbranched-chain metabolites and progressivefamilial infantile cerebral dysfunction. Thisoriginal documentation of branched-chainamino acid-related genetic disorders has beenexpanded by clinical observation and biochemi-cal experimentation to include the catabolicdysfunctions listed in Table 1. These syndromesrange from fulminating to inapparent. Readersinterested in more complete descriptions ofthese metabolic anomalies in man are referredto references 43 and 63. The intrinsic difficul-ties of studying catabolic pathways of low enzy-matic activity in man and the paucity of clinicalmaterial have led to the utilization of prokar-yotic metabolic models. A better understandingof these prokaryotic metabolic pathways mayyield more therapeutic regimens, resulting inalleviation of the inborn error. An example ofclinical application of knowledge obtained fromthis basic research is the current therapeuticadministration of biotin to patients sufferingfrom 3-methylcrotonylglycinuria. The functionof biotin was first elucidated by using bacterial3-methylcrotonyl coenzyme A (CoA) carboxyl-ase in Achromobacter grown on isovalerate

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AMINO ACID CATABOLISM 43

D-valineD-isoleucine +D-leucine

L-valineL-isoleucineL-leucine

2-ketoisovalerate -4-----------panltothenlate2-keto-3-methylvalerate2-ketoisocaproate

(+)-camphor------ - isobutyryl-CoA2-methylbutyryl-CoAisovaleryl-CoA

methylacrylyl-CoAtiglyl-CoA 3-hydroxy-

geraiiiol & Jarnesol------- ------*3-methylcrotonyl-CoA isobu ty - 3is

3-methylgl utacony l-CoA 2-methyl-3-hydroxy -3-methylglutaconylCo

butyryl-CoA

mnealonate'------------------------------ 3-hydroxy-3-methylglutaryl -CoA 2-methylpaetoacetyl-CoA

propionyl-CoA +acetoaetate -tacetyl-CoA

1-hydroxy-sobutyrate

methyl malonatesemialdehyde

r-opionyl-CoA

acetyl-CoA

FIG. 1. Pathway for the catabolism of D- and L-branched-chain amino acids in bacteria.

(20). Some types of ketoacidemias may be simi-larly ameliorated by thiamine and vitamin B....

ENZYMES COMMON TO CATABOLISMOF ALL THREE BRANCHED-CHAIN

AMINO ACIDSThe complete catabolism of each branched-

chain amino acid requires the cooperation oftwo sequential series of reactions. The enzymesin the first series comprise a common pathwaycatalyzing the conversion of isoleucine, leucine,and valine to their respective acyl-CoA deriva-tives. The overlapping specificities inherent inthis arrangement are advantageous to an orga-nism. Metabolic redundancy is avoided since asingle structural gene can control the catabo-lism of closely related analogues. However,branched-chain metabolites formed subsequentto this common pathway are catabolized bythree separate enzyme series, one specific foreach amino acid. Several other organic com-pounds, such as terpenes and long-chain alco-hols, are degraded to metabolites identical tothose found in amino acid-specific pathways.One advantage of separate enzyme series isthat intermediates from converging pathways

TABLE 1. Biochemical defects in genetic disorders ofbranched-chain amino acid catabolism

Disorder Biochemical defect

Maple syrup urine disease (5 Branched-chain keto acidvariants) dehydrogenase

Hypervalinemia Valine transaminaseIsovaleric acidemia Isovaleryl-CoA dehydro-

genase3-Methylcrotonylglycinuria 3-Methylcrotonyl-CoA

carboxylaseMethylmalonic acidemiaType I Methylmalonyl-CoA race-

maseType II Methylmalonyl-CoA car-

bonylmutaseType III 5-Deoxyadenosyl cobala-

min synthesisType IV Vitamin B,2 formation

(sulfur amino acid me-tabolism)

2-Methyl-3-hydroxybutyric 2-Methylacetoacetyl-CoAaciduria thiolase

do not gratuitously induce nonessential en-zymes of the common pathway.This section will discuss the catabolism of

isoleucine, leucine, and valine in a commonpathway by branched-chain amino acid trans-

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44 MASSEY, SOKATCH, AND CONRAD

aminase, )-amino acid dehydrogenase, andbranched-chain keto acid dehydrogenase. Al-though suggested by some data, there is insuffi-cient evidence to make a categorical assign-ment of acyl-CoA dehydrogenase and enoyl-CoA hydratase to this common pathway. Thephysiological role of L-amino acid oxidase iseven more obscure, although its end productwould be metabolized via this common path-way.

Branched-Chain Amino Acid TransaminaseThe ability to transfer an amino group be-

tween amino acid and keto acid is a metabolicnecessity for both prokaryotic and eukaryoticorganisms. Transamination is unique amongenzymatic processes in that it is equally impor-tant in both biosynthesis and catabolism ofamino acids.

Branched-chain amino acid transaminase(branched-chain amino acid 2-ketoglutarateaminotransferase; EC 2.6.1.42) was first de-scribed by Rudman and Meister (56), who sepa-rated two enzymes in Escherichia coli on thebasis of their ability to transaminate differentamino acids with 2-ketoglutarate. Transami-nase A was most active with aromatic aminoacids and oxalacetate, whereas transaminase Bpreferred branched-chain amino acids and2-ketoglutarate acid.Norton and Sokatch (37) purified branched-

chain amino transaminase (transaminase B)from Pseudomonas aeruginosa. The enzymewas active with a number of amino acids over awide pH range and appeared to be synthesizedconstitutively. Enzyme activity was limited toLisomers. The Michaelis constants for aminoand keto acid substrates were consistent with apossible dual function in both biosynthesis andcatabolism. Voellmy and Leisinger (68) haverecently reported such a dual-function trans-aminase. N2-acetylornithine 5-aminotrans-ferase from P. aeruginosa functions in botharginine catabolism and biosynthesis.Martin et al. (33) isolated a P. putida mutant

that was unable to catabolize valine, leucine, orisoleucine. However, the branched-chain ketoacids supported growth. The mutant was not anamino acid auxotroph. A reduced level ofbranched-chain amino acid transaminase activ-ity was the only discernible enzymatic differ-ence between this pleiotropic mutant and thewild type. It was not determined whether theactivity observed was due to a mutation in oneof multiple transaminases with overlappingspecificities or to alteration of a single bifunc-tional transaminase, with loss of only the cata-bolic activity. It is noteworthy that neitherMartin et al. (33) nor Marinus and Loutit (29)

were able to isolate Pseudomonas mutantscompletely devoid of branched-chain aminoacid transaminase activity.Puukka et al. (48) reported that branched-

chain amino acid transaminase in P. fluores-cens was inducible by combinations ofbranched-chain amino acids or their keto acids.They did not ascertain the mechanism for regu-lation of enzyme synthesis.Coleman et al. (7, 8) purified branched-chain

amino acid transaminases from Salmonella ty-phimurium and S. montevideo. No significantkinetic differences were found between the en-zymes of the two Salmonella species. Detailedkinetic analysis indicated that transaminationproceeded via a binary "ping-pong" mechanism.Transaminase mutants were isolated from Sal-monella by Kiritani (23). He observed normalwild-type growth rates only when the minimaimedium was supplemented with all threebranched-chain amino acids. This phenomenonmost likely reflects the biosynthetic rolebranched-chain transaminase plays in Salmo-nella metabolism.

L-Amino Acid Oxidase

Bernheim et al. (3) observed in 1935 thatProteus vulgaris cell suspensions oxidized mostof the naturally occurring L-amino acids, in-cluding the branched-chain amino acids.Stumpf and Green (65) reported that the en-zymes involved in the oxidation of 22 aminoacids could be classified by differences of invitro enzymatic stability. The most stable oxi-dase attacked only the L-isomers of the aminoacids, producing the keto acid, ammonia, andwater.Two distinct particulate L-amino acid oxi-

dases (L-amino acid:oxygen oxidoreductase[deaminating]; EC 1.4.3.2) were separated fromProteus rettgeri by Duerre and Chakrabarty(12). The substrate specificity of one enzymewas limited to basic amino acids and citrulline.The other oxidase had a broader specificity,catalyzing the oxidative deamination of mon-oaminomonocarboxylic, imino, aromatic, sul-fur-containing, and 3-hydroxy,L-amino acids.The in vivo role of L-amino acid oxidases in

bacterial branched-chain amino acid catabo-lism is unknown. Stumpf and Green (65) de-tected L-amino acid oxidase in only three of tengenera they examined, indicating a limited dis-tribution. Similarly, Coudert and Vandecas-teele (11) surveyed several species and found L-amino acid oxidase activity in Corynebacter-ium, Brevibacterium, Micrococcus, and Myco-bacterium, but not in P. putida, E. coli, orBacillus subtilis. The oxidase from Corynebac-

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terium was unusual since it was the only bacte-rial oxidase reported to produce hydrogen per-oxide rather than water. Several L-amino acidsinduced the oxidase activity. These results im-ply that L-amino acid oxidase is neither ubiqui-tous nor essential to the metabolism of all bac-teria.

1-Amino Acid DehydrogenaseAnother method of enzymatically producing

branched-chain keto acids is via the oxidativedeamination of 1-stereoisomers of the aminoacids. In bacteria, this reaction is catalyzed by1)-amino acid dehydrogenase (EC 1.4.99.1). Pro-karyotic )-amino acid deamination systemsthus far reported differ mechanistically fromtheir eukaryotic counterparts in that they uti-lize 0.5 mol of oxygen per mol of amino acidoxidized, implying that water rather than hy-drogen peroxide is the final product.

Early studies ofbacterial 1)-amino acid oxida-tion were made by Bernheim et al. (3) and byStumpf and Green (Fed. Proc. 5:I157, 1946) withcell-free extracts from Proteus. Both groups re-ported utilization of 1 atom of oxygen per mol ofamino acid oxidized. However, similar sto-ichiometry would result from the action of cata-lase on peroxide if both are present. Sokatchand his colleagues (31, 36) found that P. aerugi-nosa 1)-amino acid dehydrogenase preparationswere free of catalase. Neither Yoneya andAdams (73) nor Stumpf and Green (65) wereable to detect peroxide formation in their stud-ies. By contrast, kidney -amino acid oxidase(EC 1.4.3.3) consumes 1 mol of oxygen per molof amino acid oxidized and forms peroxide as anend product.Tsukada (66) partially purified two distinct '-

amino acid dehydrogenases from P. fluorescens.One species was synthesized constitutively andhad an absolute specificity for 2,6-dichloroindo-phenol as the electron acceptor. The other en-zyme was induced by growth on -tryptophanand was specific for methylene blue as the elec-tron acceptor. Both dehydrogenases contained aflavin adenine mononucleotide (FAD) pros-thetic group and were active with a number of1-isomers, including those of the branched-chain amino acids. Both enzymes were inactivewith acidic amino acids. The two dehydroge-nases were dissimilar in substrate specificity,electron acceptors, thermal lability, and induc-ibility, but had similar pH optima, absorptionspectra, and enzyme kinetics. Neither enzymewas affected by the presence of divalent cationsand chelating agents.Norton and Sokatch (36) reported that cell-

free extracts of P. aeruginosa grown on DL-

valine catalyzed the oxidative deamination ofn-valine to 2-ketoisovalerate. Enzyme activitywas inducible and consumed 1 atom of oxygenper mol of keto acid produced. This line of in-vestigation was continued by Marshall and So-katch (31), who achieved a 13-fold purificationof 1)-amino acid dehydrogenase. The partiallypurified enzyme preparation was active withthe -isomers of branched-chain, aromatic, andbasic amino acids, but was inactive with acidicamino acids. Enzyme activity was exceedinglyunstable and particulate in nature. The pHoptimum varied as a function of the substrate.Comparative studies of the bleaching of D-

amino acid dehydrogenase indicated the pres-ence of cytochrome c and flavin. Biochemicalexperimentation and genetic analysis by So-katch and his associates (32, 33) indicated thatpseudomonal 1)-amino acid dehydrogenase is aregulatory segment separate from the otherbranched-chain amino acid catabolic enzymes.Yoneya and Adams (73) described an induc-

ible allohydroxy-D-proline oxidase from Pseu-domonas striata. Strong similarities werenoted between this enzyme and previously de-scribed -amino acid dehydrogenases in absorp-tion spectra, stoichiometry of oxygen consump-tion, particulate nature, broad substrate speci-ficity, and induction by -amino acids.The presence of -amino acid deamination

enzymes raises questions about the role of theenzymes in bacterial metabolism. Presumably,functional enzymes exist because they are, orwere, of survival benefit to the organisms. Onemight logically hypothesize that 1)-amino aciddehydrogenases evolved to aid the catabolism ofthe mucopeptide layer of bacterial cell walls.However, this premise is contraindicated by theobservation that bacterial 1-amino acid dehy-drogenases characterized so far are conspicu-ously inactive with -glutamate, a major com-ponent of the murein sacculus. Another para-doxical aspect of -amino acid dehydrogenasesis their characteristic broad substrate specific-ity, which includes certain amino acids, the -

isomers of which are not known to occur innature. The physiological function of -aminoacid dehydrogenase remains speculative.

Branched-Chain Keto Acid DehydrogenaseIn bacteria, branched-chain keto acid dehy-

drogenase appears to be an enzyme complexthat oxidatively decarboxylates all threebranched-chain keto acids to their respectiveacyl-CoA derivatives. In contrast, Connelly etal. (4, 9) have reported that mammalian sys-tems utilize two dehydrogenases: one specificfor 2-ketoisovalerate (EC 1.2.4.4) and the other

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acting on 2-keto-3-methylvalerate and 2-keto-isocaproate (EC 1.2.4.3).Rudiger et al. (55) reported that partially

purified branched-chain keto acid dehydrogen-ase in Streptococcus faecalis was a multi-en-zyme complex similar to pyruvate and 2-keto-glutarate dehydrogenases. They demonstratedthat the complex contained three different en-zymes participating in the oxidation ofbranched-chain keto acids: decarboxylase,transacylase, and flavin lipoamide oxidoreduc-tase. Sasaki (58) partially purified an enzymefrom Proteus vulgaris that catalyzed the decar-boxylation of branched-chain keto acids andrequired CoA and nicotinamide adenine dinu-cleotide (NAD) as cofactors. Partially purifiedbranched-chain keto acid dehydrogenase com-plexes from Bacillus subtilis (35) and S. fae-calis (55) oxidized all three branched-chain ketoacids. Although all three branched-chain ketoacids served as substrates, they were notequally active. Both bacterial enzymes pre-ferred 2-ketoisovalerate and were least activewith 2-ketoisocaproate. In contrast, cell-free ex-tracts of P. putida most efficiently oxidized 2-keto-3-methylvalerate (30). The addition of 1mM L-leucine, or L-isoleucine but not L-valine,caused a 3.5-fold increase in activity when 10mM 2-ketoisovalerate was used as substrate.

Willecke and Pardee (71) isolated and de-scribed a B. subtilis mutant that was defectivein branched-chain keto acid dehydrogenase.This mutant required a short branched-chainfatty acid derived from either leucine, isoleu-cine, or valine for growth. Their results sug-gested that in B. subtilis a single enzyme wasresponsible for the oxidative decarboxylation ofall three branched-chain fatty acids. Namba etal. (35) found that the B. subtilis enzyme wasconstitutive, an observation consistent with itsprobable role in lipogenesis.Martin et al. (33) characterized P. putida

mutants that showed a simultaneous loss ofability to oxidize all three keto acids, indicatingthat one enzyme complex was involved in theoxidation of all three branched-chain aminoacids.

In P. putida, the inducers of the dehydrogen-ase have been identified by Marshall and So-katch (32) as the branched-chain keto acidsrather than the amino acids. A mutant defi-cient in the production of keto acids from L-branched-chain amino acids also lacked dehy-drogenase activity unless grown in the presenceof at least one branched-chain keto acid. In P.putida the three keto acids were equally effec-tive as inducers of branched-chain keto aciddehydrogenase.

Branched-Chain Acyl-CoA DehydrogenaseLittle is known about branched-chain acyl-

CoA dehydrogenase in either prokaryotes oreukaryotes. Marshall and Sokatch (32) reporteda constitutive, very low activity for isobutyryl-CoA dehydrogenase in extracts of P. putida.Only minor differences in activity were ob-served when either butyryl-CoA or isobutyryl-CoA was used as substrate. They did not deter-mine whether the enzyme was specific forbranched-chain acyl groups or was an expres-sion of butyryl-CoA dehydrogenase (EC1.3.99.2) activity in the beta oxidation of fattyacids. Engel and Massey (13) purified butyryl-CoA dehydrogenase from Peptostreptococcuselsdenii but only assayed with straight-chainacyl-CoA derivatives, so the enzyme's role inbranched-chain catabolism is unknown.

ENZYMES SPECIFIC FOR INDIVIDUALAMINO ACID PATHWAYS

LeucineThree enzymes beyond the common pathway

are required to complete the catabolism of leu-cine to acetoacetate. These leucine-specific en-zymes are 3-methylcrotonyl-CoA carboxylase(EC 6.4.1.4), 3-methylglutaconyl-CoA hydra-tase (EC 4.2.1.18), and 3-hydroxy-3-methyl-glutaryl-CoA lyase (EC 4.1.3.4).Lynen et al. (25) first demonstrated the car-

boxylation of 3-methylcrotonyl-CoA in extractsof Mycobacterium and Achromobacter. Thesebacteria were isolated from soil by using isoval-erate as the sole source of carbon. Rilling andCoon (52) demonstrated the carboxylation of 3-methylcrotonyl-CoA in extracts of Pseudomo-nas oleovorans.The biochemical function of biotin was eluci-

dated by Himes et al. (20) through their studiesof 3-methylcrotonyl-CoA carboxylase from Ach-romobacter. When the purified enzyme was in-cubated with adenosine 5'-triphosphate (ATP)and radioactive bicarbonate under appropriateconditions, a labeled carboxylated enzyme re-sulted. The enzyme complex then catalyzed thetransfer of the carbonyl moiety to 3-methylcro-tonyl-CoA, forming 3-methylglutaconyl-CoA.Massey et al. (34) demonstrated that 3-methyl-crotonyl carboxylase in P. putida was inducibleby growth on isovalerate as the sole carbonsource. 3-Methylglutaconyl-CoA was enzymati-cally hydrated to 3-hydroxy-3-methylglutaryl-CoA. This hydration has also been reported inMycobacterium (19) and Achromobacter (25).The final enzyme specific for leucine catabo-

lism, 3-hydroxy-3-methylglutaryl-CoA lyase,has been reported in an actinomycete grown on

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mevalonic acid (61) and has been partially puri-fied from P. putida (Massey and Sokatch, un-published data).Both 3-methylcrotonyl-CoA carboxylase and

3-hydroxy-3-methylglutaryl-CoA lyase were in-duced in P. putida grown on isovalerate. Theaddition of glucose or glutamate to cells grow-ing on DL-leucine repressed the synthesis ofboth the carboxylase and lyase (34).Winnacker and Barker (72) studied the me-

tabolism of acetoacetate in P. putida. Theydemonstrated the presence of acetoacetyl-CoA:succinyl CoA transferase (EC 2.8.3.5) andacetoacetyl-CoA thiolase (EC 2.3.1.9) in crudeextracts of cells grown on 3-amino-n-butyrate.The combined action of these enzymes com-pleted the degradation of acetoacetate to acetyl-CoA and acetate. Their observations that isocit-rate lyase and malate synthetase were inducedduring growth on 3-amino-n-butyrate led themto hypothesize that the resultant acetyl-CoAwas metabolized via the glyoxylate cycle.

Pauli and Overath (45) reported a similarpathway of acetoacetate degradation in E. coli.The addition of acetoacetate to growth mediumresulted in a 3,000-fold increase of acetoacetyl-CoA:succinyl-CoA transferase and acetoacetyl-CoA thiolase. The structural genes for theseenzymes were closely linked to a regulatorygene.

IsoleucineThe bacterial oxidation of tiglyl-CoA to ace-

tyl-CoA and propionyl-CoA was first describedby Conrad et al. (10) in P. putida. Tiglyl-CoAhydratase and 2-methyl-3-hydroxybutyryl-CoAdehydrogenase were partially purified andcharacterized. The synthesis of these two en-zymes and 2-methylacetoacetyl-CoA thiolasewas induced by growth on either isoleucine, 2-keto-3-methylvalerate, 2-methylbutyrate, ortiglate. Inductive and kinetic analyses indi-cated that the catabolic hydratase and dehydro-genase were distinguishable from their beta-oxidative counterparts and are unique to thecatabolism of isoleucine in P. putida. Enzymeactivity was limited to 2-methyl-3-hydroxy-butyryl-CoA, 3-hydroxybutyryl-CoA, and 2-hydroxy-3-methylvaleryl-CoA. The purified de-hydrogenase had an absolute requirement forNAD and CoA esters. The enzymatic produc-tion of 2-methylacetoacetyl-CoA was authen-ticated by deacylation, chemical decarboxyla-tion, and identification of methylethyl ketonehydrazone by thin-layer chromatography.

ValineAt least two enzymatic reactions, and per-

haps as many as four, are known to be specific


for the conversion of methylacrylyl-CoA to pro-pionyl-CoA.The first valine-specific enzymatic conver-

sion is the hydration ofmethylacrylyl-CoA to 3-hydroxyisobutyryl-CoA. Puukka (46) reportedthat enoyl-CoA hydratase (EC 4.2.1.17) in P.fluorescens was induced by growth onbranched-chain amino acids, branched-chain 2-keto acids, and short branched-chain fattyacids.There is still no conclusive evidence that

methylacrylyl-CoA hydratase (enoyl-CoA hy-dratase) is unique to the valine catabolic path-way. A hydration step is required for the catab-olism of every branched-chain amino acid.Puukka (46) reported that isoleucine and leu-cine catabolic intermediates were more effec-tive than valine as inducers of the hydratasewhen used as the sole source of carbon. Thespecificity of enoy*-CoA hydratases is discussedlater in this review.3-Hydroxyisobutyryl-CoA is enzymatically

deacylated to free 3-hydroxyisobutyrate andCoA. Nurmikko et al. (41) and Marshall (30)have described this activity in Pseudomonas.Nurmikko et al. (41) reported that 3-hydroxy-isobutyryl-CoA hydrolase (EC 3.1.2.4) was in-duced during the growth of P. fluorescens oneither valine, 2-ketoisovalerate, isobutyrate, or3-hydroxyisobutyrate. Isobutyrate and 3-hy-droxyisobutyrate were the most effective in-ducers. Several Krebs cycle intermediates re-pressed the formation of the hydrolase. ThLuniqueness of 3-hydroxyisobutyryl-CoA hydro-lase to valine catabolism has not been deter-mined.Puukka and Nurmikko (50) published studies

on another valine-specific enzyme, 3-hydroxy-isobutyrate dehydrogenase (3-hydroxyisobutyr-ate:NAD oxidoreductase; EC 1.1.1.31). The pat-terns of induction and repression for this en-zyme and for 3-hydroxyisobutyryl-CoA hydro-lase were similar. Bannerjee et al. (2) purified3-hydroxyisobutyrate dehydrogenase 85-foldfrom P. aeruginosa grown on valine. The en-zyme seemed to be specific to valine catabolisinsince, of a number of analogues tested, 3-hy-droxyisobutyrate was the only substrate oxi-dized.Methylmalonate semialdehyde is oxidatively

decarboxylated to propionyl-CoA and carbondioxide by methylmalonate semialdehyde de-hydrogenase (EC 1.2.1.27), which was firstcharacterized by Sokatch et al. (62) in extractsofP. aeruginosa grown on DL-valine. They puri-fied the enzyme to a form homogenous by discgel electrophoresis and analytical ultracentrif-ugation (2). The purified enzyme catalyzed theoxidation of either methylmalonate semialde-


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hyde or propionaldehyde, concurrently withacylation of these substrates by CoA. The in-duction of methylmalonate semialdehyde dehy-drogenase was studied in P. fluorescens byPuukka et al. (47) and in P. putida by Marshalland Sokatch (32). The enzyme was induced byeither valine, 2-ketoisovalerate, isobutyrate, or3-hydroxybutyrate. Kinetic analysis by Mar-shall and Sokatch (32) of 3-hydroxyisobutyratedehydrogenase and methylmalonate semialde-hyde dehydrogenase induction indicated thattheir synthesis was coordinately controlled.

SPECIFICITY OF ENOYL-CoAHYDRATASES

Although extensively studied in eukaryoticorganisms, the physiological role of enoyl-CoAhydratases in prokaryotic catabolic pathwaysremains relatively unexplored. Our discussionwill be limited to catabolism and excludes theenoyl-CoA hydratases of lipid biosynthesis,which have been discussed in other reviews(22). This may be an artificial division, sincedata clearly delineating the anabolic and cata-bolic hydratases are lacking. This qualificationis particularly noteworthy since the carbonskeletons of all three branched-chain aminoacids are incorporated in toto into branchedlong-chain fatty acids in a number of bacterialspecies, including B. subtilis (21) and Micrococ-cus lysodeikticus (M. G. Macfarlane, Biochem.J. 79:4P, 1961).Conrad et al. (10), during a study of isoleu-

cine catabolism in P. putida, partially purifiedand characterized a hydratase catalyzing thehydration of tiglyl-CoA (2-methylcrotonyl-CoA)and crotonyl-CoA. The corresponding enoyl-CoA derivative from leucine catabolism, 3-methylglutaconyl-CoA, was not hydrated, sug-gesting that P. putida requires at least twohydratases to catabolize branched-chain aminoacids. Increased tiglyl-CoA hydratase activitywas found in cells grown in the presence ofisoleucine or isoleucine catabolic intermedi-ates. Valine and leucine were slightly induc-tive. Conrad also noted that extracts of croton-ate-grown cells had increased levels of crotonyl-CoA hydratase without a corresponding in-crease in the level of tiglyl-CoA hydratase.These data implied that the hydration of cro-tonyl-CoA and tiglyl-CoA is catalyzed by sepa-rate inducible hydratases possessing mixedsubstrate specificities. Thus P. putida croton-ase activity was limited to crotonyl-CoA, buttiglyl-CoA hydratase was active with both cro-tonyl-CoA and tiglyl-CoA.Puukka's (46) investigations of valine catabo-

lism by P. fluorescens documented induction of

methylacrylyl-CoA hydratase by growth onbranched-chain amino acids, branched-chain 2-keto acids, and short branched-chain fattyacids. Highest levels of enoyl-CoA hydratasewere detected when the carbon source was iso-leucine or 2-ketoisocaproate. The specificity ofthe enzyme remains uncertain since methyl-acrylyl-CoA was the only substrate tested.Waterson et al. (69) purified crotonase from

Clostridium acetobutylicum without ascertain-ing its function, if any, in branched-chainamino acid catabolism. Their studies indicatedthat crotonase was one of multiple enoyl-CoAhydratases found in crude extracts. Clostridialcrotonase was specific for crotonyl-CoA andhexenoyl-CoA. Although some molecular andcatalytic homologies with bovine crotonase sug-gest a distant phylogenic relationship, evolu-tionary pressures have resulted in enzymeswith distinct substrate specificities.Weeks et al. (70) described an enoyl-CoA hy-

dratase in E. coli that was active with crotonyl-CoA. The hydratase was coordinately inducedwith beta oxidation enzymes when the cellswere grown on long-chain fatty acids. Croton-ase induction was not observed in amino acidmedium, which suggests that its activity in E.coli is limited to beta oxidation of fatty acids.

Seubert and Fass (59, 60) enriched isohexen-ylglutaconyl-CoA hydratase (EC 4.2.1.57) 100-fold from Pseudomonas citronellolis. Theyfound that this enzyme functioned in the catab-olism of long branched-chain alcohols. The en-zyme was active with unsaturated dicarboxylicacid derivatives of geraniol and farnesol.The available data suggest that the enoyl-

CoA hydratases functioning in catabolism ofbranched-chain acids are probably distinct en-zymes from the anabolic hydratases active inlipid biosynthesis. The Pseudomonas catabolicenoyl-CoA hydratases thus far reported haveall been inducible.

CATABOLIC PATHWAYS CONVERGINGWITH BRANCHED-CHAIN AMINO ACID

PATHWAYSGeraniol and Farnesol

Seubert and Fass (59, 60) described an inter-esting variation of the oxidation of branched-chain compounds. Geraniol and farnesol arelong branched-chain alcohols that can be de-graded by one species ofPseudomonas (Fig. 2).The catabolic pathway of these alcohols resem-bles beta oxidation, with additional carboxyla-tion and cleavage steps similar to leucine catab-olism. After conversion of geraniol and farnesolto the acyl-CoA derivatives, the methyl sidechain of the resultant enoyl-CoA was carbox-

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H3C OH H3CW-~ HO H3HCOHCH3 CH3 CH3 C

3 CH3 CH3 CH3

FARNESOLFIG. 2. Long branched-chain alcohols

ylated. This was followed by hydration of thedouble bond. Seubert and Fass found that thepurified enoyl-CoA hydratase was active with3-methylcrotonyl-CoA as well as long-chain de-rivatives of geranyl-CoA and farnesyl-CoA.The cleavage of the hydroxylated CoA deriva-tives to acetate and a 3-ketoacyl-CoA derivativewas catalyzed by hydroxyisohexenylglutaryl-CoA:acetate lyase (EC 4.1.3.26). The resultant3-ketoacyl-CoA was metabolized by a pathwayanalogous to beta oxidation. Cyclic repetition ofthe carboxylation, hydration, and cleavage re-

actions eventually resulted in the production of3-methylcrotonyl-CoA. The purified lyase de-scribed by Seubert and Fass only cleaved thehydroxy derivatives formed from geranyl-CoAand farnesyl-CoA. A separate lyase, hydroxy-methylglutaryl-CoA:acetyl-CoA lyase (EC4.1.3.4), is responsible for the cleavage of 3-hydroxy-3-methylglutaryl-CoA to acetyl-CoAand acetoacetate. The catabolic pathways forgeraniol and farnesol were induced by citronel-lol, another branched-chain alcohol (Fig. 2).

PantothenateGoodhue and Snell (16) studied the bacterial

metabolism of pantothenate by PseudomonasP-2. They found that the incomplete oxidationof pantothenate resulted in the production ofdetectable amounts of beta-alanine, pantoate,valine, and 2-ketoisovalerate in culture fil-trates. They explained the presence of theseproducts by the hydrolysis of pantothenate tobeta-alanine and pantoate, which was subse-quently converted to 2-ketoisovalerate. Theysubstantiated this pathway (Fig. 3) by partiallypurifying and characterizing the four necessary

enzymes: pantothenate aminohydrolase, EC3.5.1.22 (42); D-pantoate NAD oxidoreductase,EC 1.1.1.106 (17); i)-aldopantoate dehydroge-nase, EC 1.2.1.33 (26); and dimethylmalate:NAD oxidoreductase (decarboxylating), EC1.1.1.84 (26).Mantsala, Nurmikko, and others studied the

regulation of pantothenate catabolism, includ-ing each of the reactions shown in Fig. 3, in P.fluorescens P-2 (27, 28, 38-40). They concludedthat these four enzymes of pantothenate catab-olism and pantothenate permease were coordi-nately induced by pantoate. All of the panto-thenate catabolic enzymes were subject to

'ERANIOL CITRONELLOL'?tabolized by Pseudomonas citronellis.

CH OH 03 11

HO-CH2-C-CH-C-NH-CH2CH2COOH

C H PANTOTHENIC3 ACID

..-

tH20CH OH

31IHO-CH-C -CH-COOH2

CH3PANTOIC ACID

+ HNCHCH2COOHp-ALANINE

0 CH3OH\\H- C-C -CH-COOH

CH3 ALDOPANTOIC ACID

IC H3 OH

HOOC- C-C-COOH

CH 3 3-DIMETHYLMALIC ACID

CO2-A RCH-CH-C-COOH3

CH3 cx-KETOISOVALERIC ACIDFIG. 3. Pathway for the catabolism ofpantothenic

acid in Pseudomonas.

repression by 2-keto acids and Krebs cycle in-termediates.The 2-ketoisovalerate formed from panto-

thenate was further metabolized in P. fluores-cens P-2 by enzymes common to the catabolismof valine.

Mevalonate and 3-Hydroxy-3-MethylglutarateSiddiqi and Rodwell (61) described the metab-

olism of mevalonate to 3-hydroxy-3-methylglu-taryl-CoA in an actinomycete. Mevalonate was

acylated and then oxidized to 3-hydroxy-3-methylglutaryl-CoA by a soluble NAD-requir-ing enzyme. Manometric experiments deter-mined that at least one enzyme of mevalonatemetabolism was induced by growth on meva-

lonate. Fimognari and Rodwell (14) partiallypurified mevalonate:NAD oxidoreductase (CoAacylating) (EC 1.1.1.34) from Pseudomonas Ml

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and Mycobacterium. Their kinetic analysis in-dicated that substrate binding to the enzymerequired the presence of a carboxyl, a 3-methyl,and a 3-hydroxy group. Compounds with thisconfiguration acted either as substrate or com-petitive inhibitor.Ahmad and Siddiqi (1) isolated a pseudo-

monad capable of growing on 3-hydroxy-3-methylglutarate as the sole carbon source. Ex-tracts of cells grown on 3-hydroxy-3-methylglu-tarate were acylated in the presence of ATP,CoA, and Mg2+. The cell extracts did not trans-acylate 3-hydroxy-3-methylglutarate with suc-cinyl-CoA. Cells grown on 3-hydroxy-3-methyl-glutarate contained 3-hydroxy-3-methylglu-taryl-CoA lyase and oxidized acetoacetate, sug-gesting that the CoA derivative was catabolizedby the appropriate leucine-specific enzymes.

CamphorCamphor, a terpene, can be oxidized by some

strains of P. putida to isobutyrate. Rheinwaldet al. (51) demonstrated that the initial oxida-tive cleavage of camphor was catalyzed by en-zymes coded on plasmid-borne genes, whereasisobutyrate catabolic enzymes were coded onchromosomal genes. Therefore, the completeoxidation of camphor by pseudomonads re-quires the participation of both plasmid-borneand chromosomal genetic segments. The cam-phor plasmid was readily accepted by mostpseudomonads capable of using isobutyrate assole carbon source. The isobutyrate portion ofcamphor was then catabolized by the valine-specific enzymes.

INHIBITION OF GROWTH BYBRANCHED-CHAIN AMINO ACIDS

The growth-inhibitory effects of amino acidsincorporated into the nutritional media of het-erotrophs were first noted in Gladstone's stud-ies of Bacillus anthracis (15). Since that time(1939) numerous other analogous inhibitionshave been documented. The first insight intothis paradox of inhibition by essential nutrientswas provided by Umbarger's observation (67)that the first enzyme unique to isoleucine bio-synthesis was inhibited by the end product. Thenow familiar phenomenon of end product inhi-bition has been shown to be a ubiquitous regu-latory mechanism in most biosynthetic path-ways. Umbarger's extensive investigations ofthe molecular basis of branched-chain aminoacid inhibitions in enteric bacteria have beenresponsible for much of the current understand-ing of these regulatory relationships. However,the regulation of branched-chain amino acidbiosynthesis has been adequately reviewedelsewhere (67), and our review will be confined

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to inhibitions of growth observed whenbranched-chain amino acids are used as a solesource of carbon. This stipulation limits thediscussion primarily to the pseudomonads,whose nutritional versatility enables them tocatabolize a diverse array of natural and syn-thetic organic compounds. However, this meta-bolic versatility presents the cell with a regula-tory dilemma when the sole source of carbonand energy can also function as a regulatoryeffector, essential nutrient, or metabolite inother pathways. The metabolism of branched-chain amino acids is a good case in point, sincethe branched-chain keto acids are both the firstcatabolic and last anabolic intermediates.These dual functions of keto acids raise thepossibility of competition between anabolic andcatabolic pathways. The diverging branched-chain amino acid biosynthetic and catabolicpathways are the epitome of cellular economythrough utilization of common enzymes withoverlapping specificities and shared metabo-lites. Generally speaking, this metabolic ar-rangement in a biological system is highly de-sirable since it eliminates unnecessary duplica-tion of protein biosynthesis. However, this di-vergence may work to the detriment of the cellwhen excess end products, such as branched-chain amino acids, negate the synthesis of es-sential nutrients by repression, feedback inhi-bition, shunting of intermediates into catabolicpathways, or a combination thereof.

Inhibition of growth of P. putida by leucinehas been previously reported (33, 34). Therewas a fourfold difference in growth rates ob-tained on the L- and D-isomers (20-h generationtime versus 5 h). The metabolic basis for thisdisparity has not been elucidated, but possiblyhas its roots in a nutritionally induced upset ofdelicately balanced ratios of intermediates suchas keto acids. The keto acid of leucine, 2-keto-isocaproate, is produced 50 to 100 times fasterfrom the L-isomer by aminotransferase thanfrom the D-isomer by 1-amino acid dehydroge-nase. This potential imbalance in branched-chain keto acid pools assumes relevance whenthe substrate specificity of 3-isopropylmalatesynthetase, the first enzyme unique to leucinebiosynthesis, is taken into account. Conrad andJensen (Abstr. Annu. Meet. Am. Soc. Microb-iol. P169, p. 172, 1974) found that the synthe-tase from P. putida, like that from Salmonella(24) and Hydrogenomonas (18), catalyzed thecondensation of 2-ketoisocaproate and acetyl-CoA, synthesizing a possible antimetabolite.Inhibition of growth by L-leucine was reducedby either valine or its keto acid, 2-ketoisovaler-ate. The latter is the natural substrate for 2-isopropylmalate synthetase. 2-Isopropylmalate


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synthetase from P. putida did not recognize theketo acid from isoleucine, 2-keto-3-methyl-valerate. This is consistent with the observa-tion that neither isoleucine nor its keto acidderivative relieved growth inhibition by L-leucine.

Extensive genetic mapping of Pseudomonashas delineated significant regulatory differ-ences between the pseudomonads and entericbacteria. The structural genes for the isoleu-cine-valine biosynthetic pathway in P. aerugi-nosa are scattered around the chromosome, incontrast to their juxtaposition in an operon inE. coli (29). These genetic differences do notrule out the possibility that the molecular basesfor inhibition of growth are similar in the pseu-domonads and enterics. However, these vari-ances in growth may be manifestations of dif-ferences in catabolic capability.Recent work by Calhoun and Hatfield (5)

may necessitate other interpretations of the ex-act mechanisms of branched-chain amino acidinhibition. They proposed that the biosyntheticoperon in Salmonella could be either repressedor induced by threonine deaminase (EC4.2.1.16), depending upon the binding of threo-nine deaminase to either aminoacyl-transfer ri-bonucleic acid (tRNA) or biosynthetic interme-diates. In addition, in vitro enzyme maturationcould be modulated by isoleucine and valine. Ifthis mechanism of autoregulation also func-tions in Pseudomonas, a preponderance of anyone effector could result in a slower growthrate.

REGULATION OF DIVERGENTCATABOLIC PATHWAYS

Ideally, catabolic enzymes would be synthe-sized only in order to satisfy biosynthetic orenergy demands. The degradation of panto-thenate to propionate via 2-ketoisovalerate inP. fluorescens is an example of a catabolic path-way in which all the enzymes are inducible. Aninvolved metabolic process such as this may beinduced in segments, in order to avoid synthe-sis of early enzymes when an intermediate isavailable from other sources. P. fluorescens ex-hibits sequential induction by pantoate, 2-keto-isovalerate, isobutyrate, and 2-hydroxyisobu-tyrate. This form of metabolic regulation is ap-ropos to the catabolism of rarely encounterednutrients and is consistent with the conserva-tion of energy and essential metabolites.

Marshall and Sokatch (32) reported a differ-ent regulatory pattern for valine catabolism inP. putida. Branched-chain keto acid dehydro-genase, 3-hydroxyisobutyrate dehydrogenase,and methylmalonate semialdehyde dehydro-


genase were induced by growth on valine. How-ever, branched-chain amino acid transaminaseand branched-chain acyl-CoA dehydrogenaseappeared to be synthesized constitutively. Theconstitutive nature of a branched-chain aminoacid transaminase may be appropriate if theenzyme is essential for both catabolism andbiosynthesis in P. putida. Branched-chain acyl-CoA dehydrogenase has not yet been studiedsufficiently to permit conclusions concerning itsregulation.

In this review, we have presented data thatsubstantiate the branched-chain amino acidcatabolic pathway shown in Fig. 1. This type ofbranched pathway is unusual in bacterial phys-iology, since it consists of segments of enzymescommon to the catabolism of leucine, isoleu-cine, and valine followed by three segmentswith enzymes specific for each amino acid. Theoxidation of monophenols in P. putida is alsoaccomplished by divergent catabolic pathways.Sala-Trepat et al. (57) described two dehy-drogenases from P. putida that metabolizedmonophenolic intermediates by two mecha-nisms, but produced the same end product. Thismultiplicity of responses no doubt contributesto the immense catabolic capabilities of Pseu-domonas.

There are several reasons for the recentlyrevived interest in Pseudomonas metabolism.The recognition of the opportunistic pathogen-icity and general antibiotic insensitivity of sev-eral species has given impetus to the study oftheir physiology. Comparative genetic and reg-ulatory analyses between pseudomonads andenterics have indicated fundamental differ-ences (6). Workers in the field expect that eluci-dation of these differences may give us an in-sight into basic regulatory mechanisms. Theunsurpassed metabolic versatility and amen-able genetics ofPseudomonas have made it themetabolic model in numerous investigations ofcatabolism. The study of human anomalies ofbranched-chain amino acid catabolism by bac-terial prototypes has been conducted almost ex-clusively with pseudomonads. This review hassummarized our present state of knowledge ofbacterial catabolism of branched-chain aminoacids. The knowledge and insight gained fromthese studies not only have added to a betterbasic understanding of metabolism, but alsomay be instrumental in formulating therapeu-tic relief of inborn errors of metabolism andalleviating pseudomonal disease.

LITERATURE CITED1. Ahmad, N., and M. Siddiqi. 1973. Bacterial me-

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2. Bannerjee, D., L. E. Sanders, and J. R. So-katch. 1970. Properties of purified methyl-malonyl semialdehyde dehydrogenase ofPseudomonas aeruginosa. J. Biol. Chem.245:1828-1835.

3. Bernheim, F., M. L. C. Bernheim, and M. D.Webster. 1935. Oxidation of certain aminoacids by "resting" Bacillus proteus. J. Biol.Chem. 110:165-172.

4. Bowden, J. A., and J. L. Connelly. 1968.Branched chain a-keto acid metabolism. II.Evidence for the common identity of a-keto,f-methylvaleric acid dehydrogenases. J.Biol. Chem. 243:3526-3531.

5. Calhoun, D. H., and G. W. Hatfield. 1973. Auto-regulation: a role for a biosynthetic enzymein the control of gene expression. Proc. Natl.Acad. Sci. U.S.A. 70:2757-2761.

6. Clarke, P. H., and M. H. Richmond. 1975. Ge-netics and biochemistry of Pseudomonas.John Wiley and Sons, New York.

7. Coleman, M. S., and F. B. Armstrong. 1971.Branched-chain amino acid aminotrans-ferase of Salmonella typhimurium. Biochim.Biophys. Acta 227:56-66.

8. Coleman, M. S., W. G. Soucie, and F. B. Arm-strong. 1971. Branched-chain amino-acidaminotransferase of Salmonella typhimu-rium. II. Kinetic comparison with the en-zyme from Salmonella montevideo. J. Biol.Chem. 246:1310-1312.

9. Connelly, J. L., D. J. Danner, and J. A. Bow-den. 1968. Branched-chain a-keto acid me-tabolism. I. Isolation, purification, and par-tial characterization of bovine liver a-keto-isocaproic; a-keto-f3-methylvaleric acid de-hydrogenase. J. Biol. Chem. 243:1198-1203.

10. Conrad, R. S., L. K. Massey, and J. R. Sokatch.1974. D- and L-isoleucine metabolism andregulation of their pathways in Pseudomo-nas putida. J. Bacteriol. 118:103-111.

11. Coudert, M., and J. P. Vandecasteele. 1975.Characterization and physiological functionof a soluble L-amino acid oxidase in Coryne-bacterium. Arch. Microbiol. 102:151-153.

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rial metabolism of mevalonic acid. J. Bacte-riol. 93:207-214.

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