Cloning and sequence analysis of the genes encoding the dihydrolipoamide acetyltransferase and dihydrolipoamide dehydrogenase components of the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus

Abstract

A 2641-bp EcoRI fragment of DNA that encodes the C-terminal part of the dihydrolipoyl acetyltransferase (E2) component and the dihydrolipoamide dehydrogenase (E3) component of the pyruvate dehydrogenase complex of Bacillus stearothermophilus has been cloned in Escherichia coli. Its nucleotide sequence was determined. A 705-bp truncated open reading frame was located at the 5' end of the insert which, together with the 588-bp truncated open reading frame at the 3' end of another EcoRI fragment of B. stearothermophilus DNA previously cloned and sequenced [Hawkins, C. F., Borges, A. & Perham, R. N. (1990) Eur. J. Biochem. 191,, was identified as the gene, pdhC, encoding the E2 polypeptide chain. Direct sequence analysis of the purified E2 chain confirmed that the two EcoRI fragments are adjoining in the B. stearothermophilus genome. The E3 gene, pdhD, begins just 4 bp downstream from the stop codon of the pdhC gene. The amino acid sequences deduced from the pdhC and pdhD genes correspond to proteins of 427 amino acids (E2, M , 46265) and 469 amino acids (E3, M , 49193), respectively. Both genes are preceded by potential ribosome-binding sites and the E3 gene is followed by a stemloop structure characteristic of rho-independent transcription terminators. The B. stearothermophilus E2 and E3 chains exhibit substantial sequence similarity with the corresponding subunits of other 2-0x0-acid dehydrogenase multienzyme complexes. The cloning and sequence analysis described here complete the description of the gene cluster (pdhA, B, C and D ) which encodes the B. stearothermophilus pyruvate dehydrogenase multienzyme complex.The oxidative decarboxylation of 2-0x0-acids is accomplished in most organisms by the 2-0x0-acid dehydrogenase multienzyme complexes, producing the corresponding acyl-CoA and NADH. For the pyruvate dehydrogenase (PDH) complex, the constituent enzymes that function successively in the mechanism are pyruvate dehydrogenase (El), dihydrolipoamide acetyltransferase (E2) and dihydrolipoamide dehydrogenase (E3) (for recent reviews see [I, 21).The PDH complex from the thermophilic, Gram-positive bacterium Bacillus stearothermophilus is assembled round a core of the E2 component of 60 copies of the E2 polypeptide chain organized with icosahedral symmetry [3]. Multiple cop- ies of the El (comprising two polypeptide subunits, Elcl and Elp) and E3 components are attached to this core tightly but non-covalently. The M, of the individual components as well as the morphology of the E2 core [3, 41 closely resemble those of the PDH complexes of eukaryotes. Its thermostability makes the B. stearothermophilus PDH complex a good system for studying the structure of a 2-0x0-acid dehydrogenase complex based on icosahedral symmetry, which differs from the PDH and 2-oxoglutarate dehydrogenase complexes of Escherichia coli in that the latter have octahedral E2 cores [5, 61. The sequence of the first 21 1 amino acid residues from the N-terminus of the B. stearothermophilus E2 chain has been determined [7], showing that this ace...

The Dihydrolipoamide S-Acetyltransferase Subunit of the Mitochondrial Pyruvate Dehydrogenase Complex from Maize Contains a Single Lipoyl Domain

“…3 Additionally, the possibility of a geneencoded E2 with a single lipoyl domain is not new. The E2 subunits for S. cerevisiae (35), Bacillus stearothermophilus (42), and even A. thaliana plastid (37) PDCs all contain one lipoyl domain. In this report, we extend this list to include maize, A. thaliana, and other, if not all, plant mitochondrial E2s.…”

Mitochondrial Pyruvate Dehydrogenase (Molecular Cloning of the E1[alpha] Subunit and Expression Analysis)

“…Sequences compared are the potato sequence described in this paper and the sequences from 5. stearothermophilus (Borges et al, 1990) the activity of mtPDC, we measured the activity of the enzyme in mitochondria isolated from cotyledons. These results, shown in Figure 5A, demonstrate that there is a transient increase in mtPDC activity.…”

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

“…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 …”

“…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 .…”

“…Pseudomonas fluorescens (dldh-psefl) (Benen et al, 1989); A. vinelandii (dldh-azovi) (Westphal & de Kok, 1988); P. carbinolicus (acol-pelca) ; E. coli (dldh-ecoli) (Guest, 1987); H. influenzae (dldh-haein) (HI123 I frrom the TIGR database); B. steurothermophilus (dldh-bacst) (Borges et al, 1990); B. subtilis (dldh-bacsu) (Hemila et al, 1990); S. aureus (dldh-staau) (Hemila, 1991); Huloferax volcanii, (dldh-halvo) (Vettakkorumakankav & Stevenson, 1992). dldh, dldl, and dld2 refer to the genes encoding dihydrolipoamide dehydrogenases from the pyruvate, 2-oxoglutarate, and branched chain a-ketoacid dehydrogenase complexes, respectively.…”