Cloning and sequence analysis of the genes encoding the α and β subunits of the E1 component of the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus

Abstract

A 4175-bp EcoRI fragment of DNA that encodes the a and / 3 chains of the pyruvate dehydrogenase (lipoamide) component (El) of the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus has been cloned in Escherichia coli. Its nucleotide sequence was determined. Open reading frames (pdhA, pdhB) corresponding to the Ela subunit (368 amino acids, M , 41 312, without the initiating methionine residue) and E l p subunit (324 amino acids, M , 35 306, without the initiating methionine residue) were identified and confirmed with the aid of amino acid sequences determined directly from the purified polypeptide chains. The E1p gene begins just 3 bp downstream from the Ela stop codon. It is followed, after a longer gap of 73 bp, by the start of another but incomplete open reading frame that, on the basis of its known amino acid sequence, encodes the dihydrolipoyl acetyltransferase (E2) component of the complex. All three genes are preceded by potential ribosome-binding sites and the gene cluster is located immediately downstream from a region of DNA showing numerous possible promoter sequences. The Ela and E l p subunits of the B. stearotherrnophilus pyruvate dehydrogenase complex exhibit substantial sequence similarity with the Elm and EIP subunits of pyruvate and branched-chain 2-0x0-acid dehydrogenase complexes from mammalian mitochondria and Pseudomonas putida. In particular, the El a chain contains the highly conserved sequence motif that has been found in all enzymes utilizing thiamin diphosphate as cofactor.The 2-0x0-acid dehydrogenase multienzyme complexes catalyse the oxidative decarboxylation of pyruvate, 2-0x0-glutarate and branched-chain 2-0x0 acids, releasing C 0 2 and generating the corresponding acyl-CoA and NADH. The three constituent enzymes of the pyruvate dehydrogenase (PDH) complex are pyruvate dehydrogenase (lipoamide) (El), dihydrolipoamide acetyltransferase (E2) and dihydrolipoamide dehydrogenase (E3). In the PDH complexes from Gram-positive bacteria and mitochondria, the E l components are composed of E l a and E l p polypeptide chains and the E2 components form cores consisting of 60 copies of the E2 polypeptide chains arranged with icosahedral symmetry. These cores bind multiple copies of the E l and E3 components tightly but non-covalently (for recent reviews see [I] and [2]).The E l component catalyses the decarboxylation of pyruvate and the reductive transfer of the acetyl group to a lipoic acid moiety which is covalently bound to the E2

A Mutation in the E1α Subunit of Pyruvate Dehydrogenase Associated with Variable Expression of Pyruvate Dehydrogenase Complex Deficiency

“…In additi on, the stretch of amino acids encoded by this exon and the contiguous phosphorylation site I is highly conserved both in length and in am ino acid homology when a comparison is made between the a -subunits from multiple species, both euka ryotic and prokaryotic, of the a -keto acid deh ydrogenase complexes (Fig. 6A) (10,26,(29)(30)(31)(32)(33)(34)(35)(36). Th e structure ofthis region is also intere sting.…”

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

“…Three E1␣ BCDH peptide sequences from human (20), rat (72), and Pseudomonas putida (10) cells and the E1␣ subunit of the dual PDH/BCDH complex from B. stearothermophilus (26) were aligned to identify conserved regions that could serve as candidate sequences to design corresponding PCR primers. Several regions of extended resemblance (65) were chosen, and minimal homology requirements for PCR primer design were followed as previously discussed (19,23,56).…”

“…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).…”

Characterization of PDH beta 1, the structural gene for the pyruvate dehydrogenase beta subunit from Saccharomyces cerevisiae.

“…It is not known whether the a and P subunits catalyze separate, partial reactions or whether af3 is the functional unit. Another possibility is that E1/3 plays a role in binding E1a to the E2 core (4,5), analogous to the role of protein X in anchoring E3 to the E2 core (6)(7)(8). Attempts to separate Ela and E113 with retention of enzymatic activity have been unsuccessful.…”