We cloped the structural genes for the individual subunits of the branched-chain keto acid dehydrogenase multienzyme complex on a 7.8-kilobase EcoRI-SstI restriction fragment of Pseudomonas putida chromosomal DNA by cloning into the broad-host-range vector pKT230. A direct selection system for growth on valine-isoleucine agar was achieved by complementation of P. putida branched-chain keto acid dehydrogenawe mutants. The recombinant plasmid, pSS1-1, increased expression of branched-chain ketQ acid dehydrogenase up to five times in wild-type P. putida. The complex was expressed cQnstitutively in P. putida(pSSl-l) but was inducible in Escherichia coli HB101(pSS1-1) by high valine. E. coli minicells tra'nsformed with pSS1-1 produced three polypeptides which did not match the fopr polypeptides of the purified complex. To resolve this problem, we inserted P. putida DNA from pSS1-i into pUC18 And pUC19. The pUC-derived plasmids were used as DNA templates in an E. coli transcription-translation system. Four polypeptides were produced from the pUC18-derived plasmid which had the correct molecular weights, showing that the structural genes had been cloned. Since only weak bands were produced with the pUC19-derived plasmid, the direction of transcription was established. The locations and order of all the structural genes of branched-chain keto acid dehydrogenase were located by restriction enzyme mapping.Branched-chain keto acid dehydrogenase is an enzyme common to the catabolism of valine, leucine, and isoleucine. The enzyme has been purified from mammals (25, 26) and from Pseudomonas putida (31), Pseudomonas aeruginosa (21), and Bacillus subtilis (17). In each species, it is a multienzyme complex composed of three functional subunits: El, the dehydrogenase-decarboxylase; E2, the transacylase; and E3, lipoamide dehydrogenase. Purified branched-chain keto acid dehydrogena'se from mammnals is composed of four polypeptides, the E-1 subunit being composed of two dissimilar proteins. The E2 and E3 subunits of the Pseudomonas complexes have been identified, but it was not clear whether El consisted of one or two polypeptides. P. putida and P. aeruginosa are unusual in that they possess two functionally and structurally distinct lipoamide dehydrogenases, 29). LPD-Val is the specific E3 subunit of branched-chain keto acid dehydrogenase. Mutations affecting subunits of branched-chain keto acid dehydrogenase including LPD-Val map by conjugation at a single location on the P. putida chromosomes, suggesting that the structural genes are linked (37). LPD-Glc is the E3 subunit of 2-ketoglutarate and probably pyruvate dehydrogenase and is the L-factor of glycine decarboxylase in P.putida (28,29). In Escherichia coli there is a single lipoamide dehydrogenase which functions as the E3 subunit of the pyruvate and 2-ketoglutarate dehydrogrnases (11).There is some evidence which suggests that branchedchain keto acid dehydrogenase evolved from pyruvate dehydrogenase. Lowe et al. (17) have isolated a dual-purpose keto acid dehydrogenase fro...

The genes encoding proteins responsible for activity of the El component of branched-chain-oxoacid dehydrogenase of Pseudomonas putida have been subcloned and the nucleotide sequence of this region determined. Open reading frames encoding Ela (bkdAl, 1233 bp) and EIP (bkdA2, 1020 bp) were identified with the aid of the N-terminal sequence of the purified subunits. The M , of Ela was 45158 and of E1P was 37007, both calculated without N-terminal methionine. The deduced amino acid sequences of E l a and E1B had no similarity to the published sequences of the E l subunits of pyruvate and 2-oxoglutarate dehydrogenases of Escherichia coli. However, there was substantial similarity between the El a subunits of Pseudornonas and rat liver branched-chainoxoacid dehydrogenases. In particular, the region of the Ela subunit of the mammalian branched-chain-oxoacid dehydrogenase which is phosphorylated, was found to be highly conserved in the Pseudornonas Ela subunit. There was also considerable similarity between the E1B subunits of Pseudomonas branched-chain-oxoacid dehydrogenase and human pyruvate dehydrogenase.Branched-chain-oxoacid dehydrogenase, the second enzyme in the catabolism of valine, leucine and isoleucine, catalyzes the oxidative decarboxylation of the oxoacids of these amino acids ( Fig. 1). Branched-chain-oxoacid dehydrogenase is a multienzyme complex composed of three functional components, El, the dehydrogenase-decarboxylase, E2, the transacylase and E3, lipoamide dehydrogenase. The purified complexes from Pseudomonas putida PgG2 [l] and Pseudomonas aeruginosa PA0 [2] are composed of four polypeptides. The E2 and E3 subunits have been isolated and identified [l, 21 which indicates that at least one of the remaining peptides must be the El subunit. The purified mammalian complexes also consist of four polypeptides, Ela, Elb, E2 and E3 [3, 41. There is a specific lipoamide dehydrogenase, LPDval, for branched-chain-oxoacid dehydrogenase of Pseudomonas [l, 21. In contrast, Escherichia coli produces a single lipoamide dehydrogenase which is the E3 subunit of both pyruvate and 2-oxoglutarate dehydrogenases [ 5 ] . The activity of the bacterial complex is modulated by L-valine which increases the affinity of the El subunit for the oxoacid substrates [ 1, 21. Mammalian-branched-chain-oxoacid dehydrogenase complexes are regulated by reversible phosphorylation of serine residues of the E l a subunit [6, 71. This is similar to the regulation of activity of the mammalian pyruvate dehydrogenase which also undergoes a reversible phosphorylation of serine residues of the Ela subunit [8].An oxoacid dehydrogenase complex has been isolated from Bacillus subtilis which has both pyruvate and branchedchain-oxoacid dehydrogenase activities [9]. This dual-function complex oxidizes pyruvate and provides branched-chain fatty acids for membrane phospholipids. This evidence suggested that pyruvate dehydrogenase of B. subtilis is evolving into branched-chain-oxoacid dehydrogenase and may be a model for evolution of this dehydrogenas...

Branched-chain oxo acid dehydrogenase was purified from Pseudomonas aeruginosa strain PAO with the objective of resolving the complex into its subunits. The purified complex consisted of four proteins, of Mr 36,000, 42,000, 49,000 and 50,000. The complex was resolved by heat treatment into the 49,000 and 50,000-Mr proteins, which were separated by chromatography on DEAE-Sepharose. The 49,000-Mr protein was identified as the E2 subunit by its ability to catalyse transacylation with a variety of substrates, with dihydrolipoamide as the acceptor. P. aeruginosa, like P. putida, produces two lipoamide dehydrogenases. One, the 50,000-Mr protein, was identified as the specific E3 subunit of branched-chain oxo acid dehydrogenase and had many properties in common with the lipoamide dehydrogenase LPD-val of P. putida. The second lipoamide dehydrogenase had Mr 54,000 and corresponded to the lipoamide dehydrogenase LPD-glc of P. putida. Fragments of C-terminal CNBr peptides of LPD-val from P. putida and P. aeruginosa corresponded closely, with only two amino acid differences over 31 amino acids. A corresponding fragment at the C-terminal end of lipoamide dehydrogenase from Escherichia coli also showed extensive homology. All three peptides had a common segment of eight amino acids, with the sequence TIHAHPTL. This homology was not evident in any other flavoproteins in the Dayhoff data base which suggests that this sequence might be characteristic of lipoamide dehydrogenase.

The production of two lipoamide dehydrogenases by Pseudomonus is so far unique. One, LPD-val, is the specific E3 component of the branched-chain-oxoacid dehydrogenase and the second, LPD-glc, is the E3 component of 2-oxoglutarate dehydrogenase and the L-factor of the glycine oxidation system. The objective of the present research was to determine the nucleotide sequence of the structural gene for LPD-val in order to compare its deduced amino acid structure with that of other redox-active disulfide flavoproteins. Northern blots using mRNA isolated from P. putidu grown in media with branched-chain amino acids identified a transcript of 6.2 kb which is long enough to encode all the structural genes for the complex. The nucleotide sequence of the structural gene for LPD-Val, lpdV, was determined and consists of 459 codons plus the stop codon. The open reading frame begins two bases after the stop codon for the E2 subunit and is composed of 66.3% G + C . Codon usagc is characteristic of moderately strongly expressed genes. There is a ribosome-binding site preceding the ATG start codon and a strong candidate for a rho-independent terminator at the 3' end of the reading frame. The M , of the protein encoded is 48164 and when the M , of FAD is added, the total M , is 48949, which is very close to the value of 49 000 obtained by SDS-polyacrylamide gel electrophoresis.Similarity comparisons of LPD-Val with sequences of three other lipoamide dehydrogenases showed that LPDval was somewhat more distantly related. It is probable that the lipoamide dehydrogenases and the glutathione and mercuric reductases evolved from a common ancestral flavoprotein.Branched-chain-oxoacid dehydrogenase, the second enzyme in the catabolism of valine, leucine and isoleucine [I], is a multienzyme complex composed of three subunits: El, the dehydrogenase-decarboxylase, E2, the transacylase and E3, lipoamide dehydrogenase. The complex has been purified from bovine kidney [2], Pseudomonas putidu [3], rabbit liver [4] and Pseudomonus ueruginosu [5]. In addition, an oxoacid dehydrogenase has been purified from Bacillus subtilis [6] which has activity with pyruvate and branched-chain oxoacids. All of these complexes are composed of four polypeptides including Elcl and Elfl subunits. The mammalian complexes are regulated by phosphorylation [4, 71, while the P.~eudomonus complex is allosterically regulated by L-valine I3, 51.P. putidu and P. ueruginosu contain two lipoamide dehydrogenases, LPD-Val and LPD-glc. LPD-val was so named because it is induced in media containing branched-chain amino acids as carbon sources and is the specific E3 subunit of branched-chain-oxoacid dehydrogenase, which is its only known function [5, 81. LPD-glc is found in cells grown in glucose synthetic medium and is the E3 subunit of 2-oxoglutarate dehydrogenase [9] and the L-factor of the glycine-oxidation system in P. putidu [lo]. LPD-val and LPDglc are specific for their functions and are not interchangeable. Mutants affected in IpdV, the structural gene for LPD-val, lack b...

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