Comparison of the amino acid sequences of the transacylase components of branched chain oxoacid dehydrogenase of Pseudomonas putida, and the pyruvate and 2‐oxoglutarate dehydrogenases of Escherichia coli

Abstract

The nucleotide sequence of bkdB, the structural gene for E2b, the transacylase component of branched-chainoxoacid dehydrogenase of Pseudornonas putida has been determined and translated into its amino acid sequence. The start of bkdB was identified from the N-terminal sequence of E2b isolated from branched-chain-oxoacid dehydrogenase of the closely related species, P. aeruginosa. The reading frame was composed of 65.5% G + C with 82.3% of the codons ending in G or C. There was no intergenic space between bkdA2 and bkdB. No codons requiring minor tRNAs were utilized and the codon bias index indicated a preferential codon usage. The hkdB gene encoded 423 amino acids although the N-terminal methionine was absent from E2b prepared from P. ueruginosa. The relative molecular mass of the encoded protein was 45134 (45003 minus methionine) vs 47000 obtained by SDS/polyacrylamide gel electrophoresis. There was a single lipoyl domain in E2b compared to three lipoyl domains in E2p, and one domain in E20, the transacylases of pyruvate and 2-oxoglutarate dehydrogenases of Escherichiu coli respectively. There was significant similarity between the lipoyl domain of E2b and of E2p and E20 as well as between the EI-E2 binding domains of E2b, E2p and E20. There was no similarity between the E3 binding domain of E2b to E2p and E20 which may reflect the uniqueness of the E3 component of branchedchain-oxoacid dehydrogenase of P. putida. The conclusions drawn from these comparisons are that the transacylases of prokaryotic pyruvate, 2-oxoglutarate and branched-chain-oxoacid dehydrogenases descended from a common ancestral protein probably at about the same time.Branched-chain-oxoacid dehydrogenase is a multienzyme complex which is common to the metabolism of the branched chain oxoacids which are formed from valine, isoleucine and leucine by transamination. Branched-chain-oxoacid dehydrogenase complex performs three catalytic activities. The El component is the dehydrogenase and catalyzes the decarboxylation of the oxoacid substrate and transfer of the branched-chain fatty acyl residue to E2 reducing the lipoyl prosthetic group of the latter subunit. E2 catalyzes transacylation of the fatty acyl group from the lipoyl residue of E2 to coenzyme A. E2 is the core of all of the oxoacid multienzyme complexes and binds the El and E3 components. The E3 component is a lipoamide dehydrogenase which catalyzes the oxidation of the dihydrolipoyl residue of the E2 subunit and the reduction of NAD+. The overall reaction catalyzed by Correspondence to J. R. Sokatch, Department of Biochemistry and Molecular Biology, University of Oklahoma, Health Sciences Center, P.O. Box 26901, Oklahoma City, Oklahoma, USA-73190Abhreviutions. E2b, E2p and E20 are the transacylase components of branched-chain-oxoacid, pyruvate and 2-oxoglu'tarate dehydrogenase respectively. The structural genes for the components of branched-chain-oxoacid dehydrogenase from Pseudomonas putida are hkdA1, E l i , hkdA2, EIP, hkdB, E2 and lpdV, E3 or LPD-Val. The structural genes for the...

Sequence and organization of genes encoding enzymes involved in pyruvate metabolism in mycoplasma capricolum

“…Figure 5 shows (Ho et al ., 1989), and (odpt-human) (Dah1 et al, 1987); pig (odpa-pig) (Sermon et al, 1990); mouse (odpa-mouse) (Fitzgerald et al, 1992) and (odpt-mouse) (Fitzgerald et al, 1992); rat (odba-rat) (Zhang et al, 1987), (odpt-rat) (Cullingford et a]., 1993), and (odpa-rat) (Cullingford et al, 1994); Ascarus suum (odpt-ascu) (Johnson et al, 1992) and (odpa-ascu) (Johnson et al, 1992); C. magnum (acoa-cloma) (Kruger et a]., 1994); A . eutrophus (acoaalceu) (Priefert et al, 1991); Pelobucter carbinolicus (acoa-pelca) ; K. pneumoniae (acoa-klepn) (Deng et al, 1994); bovine (odba-bovin) ; Pseudomonasputidu (odba-psepu) (Burns et al, 1988). odpa and odpt designations correspond to pyruvate dehydrogenase, whereas odba designations correspond to cy-keto acid dehydrogenase complex gene products, respectively, and the acoa designation corresponds to acetoin catabolism complexes.…”

“…9) shows clearly that the conserved residues (reverse shading) are found in the M. capricolum sequence. Boxes shaded in grey cor- laidlawii (odpb-achla) (Wallbrandt et al, 1992); B. subtilis (odpb-bacsu) (Hemila et al, 1990) and (odbb-bacsu) (Wang et al, 1993); E. stearothermophilus (odpb-bacst) (Borges et al, 1990), human (odbb-human) (Nobukuni et al, 1990a) and (odpb-human) (Ho & Patel, 1990); bovine (odbb-bovin) (Nobukuni et al, 1990b); rat (odbb-rat) (Zhao et al, 1992); P. putida (odbb-psepu) (Burns et al, 1988); A . suum (odpb-ascu) (Wheelock et al, 1991); yeast (odpb-yeast) (Miran et al, 1993); A .…”

“…capricolum (odp2-mycca) (this work); M . genitalium (odp2-mycge) (MG272 from the TlGR database); B. sieurothermophilus(odp2-bacst) (Borges et al, 1990); B. subtilis (odp2-bacsu) (Wang et al, 1993), (odb2-bacsu) (Wang et al, 1993), and (odo2-bacsu) (Carlsson & Hederstedt, 1989); C. magnum (acoc-cloma) (Kruger et al, 1994); K. pneumoniue (acoc-klepn) (Deg et al, 1994); P. carbinolicus (acoc-pelca) ; P. puiida (odb2-psepu) (Burns et al, 1988); A. vinelandii(odo2-azovi) (Westphal.3~ de Kok, 1990) and (odp2-azovi) (Hanemaaijer et al, 1988); E. coli (odo2-ecoli) (Spencer et at., 1984) and (odp2-ecoli) (Guest, 1987); Staphyfococcus a u r a s (odp2-staur) (Hemila, 1991); Neurospora crassa (odptneucr) (Kreader et al, 1989); yeast (odp2-yeast) (Niu et al, 1988) and (odo2-yeast) (Repetto …”

Cloning and sequencing of a cluster of genes encoding branched-chain alpha-keto acid dehydrogenase from Streptomyces avermitilis and the production of a functional E1 [alpha beta] component in Escherichia coli

“…In these systems, it was found that the genes encoding the BCDH complex were clustered in an operon. The genes encoding the BCDH complex of P. putida (bkd genes) and the PDH/BCDH dual complex of B. subtilis and Bacillus stearothermophilus are clustered in the sequence E1␣, E1␤, E2, and E3 (10,11,26,27,59). Recently, the genes for the branchedchain fatty acid-specific BCDH from B. subtilis were cloned and sequenced (64).…”

“…E3 (10,11,26,27,59). Recently, the genes for the branchedchain fatty acid-specific BCDH from B. subtilis were cloned and sequenced (64).…”