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Amino acid sequence analysis of the lipoyl and peripheral subunit-binding domains in the lipoate acetyltransferase component of the pyruvate dehydrogenase complex from Bacillus stearothermophilus.

ABSTRACT: The pyruvate dehydrogenase multienzyme complex from Bacillus stearothermophilus comprises a structural core, composed of 60 dihydrolipoamide acetyltransferase (E2p) subunits, which binds multiple copies of pyruvate decarboxylase (E1p) and dihydrolipoamide dehydrogenase (E3) subunits. After limited proteolysis with chymotrypsin, the N-terminal lipoyl domain of E2p was excised, purified and sequenced. The residual complex, which remained assembled, was then digested with trypsin under mild conditions. This treatment promoted complete disassembly of the complex and the various components were separated by gel filtration and h.p.l.c. A folded fragment of E2p containing about 50 amino acid residues was identified as being responsible for binding the E3 subunits, although, unlike the corresponding region of the E2p or E2o chains of the pyruvate dehydrogenase or 2-oxoglutarate dehydrogenase complexes from Escherichia coli, the fragment also bound E1p molecules. Further peptide purification and sequence analysis allowed the determination of the first 211 amino acid residues of the B. stearothermophilus E2p chain, thus providing the complete primary structure of the lipoyl domain, the E1p/E3-binding domain and the regions of polypeptide chain, probably highly flexible in nature, that link the domains to each other and to the inner-core (E2p-binding) domain. Several of the proteolytically sensitive sites were also identified. The sequence of the B. stearothermophilus E2p chain shows close homology with the sequences of the E2p and E2o chains from E. coli, although significant differences in structure are apparent. Detailed evidence for the sequence of the peptides obtained by limited proteolysis and further chemical and enzymic cleavages have been deposited as Supplementary Publication SUP 50142 (11 pages) at the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 6BQ, U.K., from whom copies may be obtained as indicated in Biochem. J. (1988) 249, 5.

SUBMITTER: Packman LC

PROVIDER: S-EPMC1149109 | biostudies-other | 1988 May

REPOSITORIES: biostudies-other

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Project description:Lipoic acid is an organosulfur cofactor essential for several key enzyme complexes in oxidative and one-carbon metabolism. It is covalently bound to the lipoyl domain of the E2 subunit in some 2-oxoacid dehydrogenase complexes and the H-protein in the glycine cleavage system. Lipoate-protein ligase (Lpl) is involved in the salvage of exogenous lipoate and attaches free lipoate to the E2 subunit or the H-protein in an ATP-dependent manner. In the hyperthermophilic archaeon Thermococcus kodakarensis, TK1234 and TK1908 are predicted to encode the N- and C-terminal regions of Lpl, respectively. TK1908 and TK1234 recombinant proteins form a heterodimer and together displayed significant ligase activity toward octanoate in addition to lipoate when a chemically synthesized octapeptide was used as the acceptor. The proteins also displayed activity toward other fatty acids, indicating broad fatty acid specificity. On the other hand, lipoyl synthase from T. kodakarensis only recognized octanoyl-peptide as a substrate. Examination of individual proteins indicated that the TK1908 protein alone was able to catalyze the ligase reaction although with a much lower activity. Gene disruption of TK1908 led to lipoate/serine auxotrophy, whereas TK1234 gene deletion did not. Acyl carrier protein homologs are not found on the archaeal genomes, and the TK1908/TK1234 protein complex did not utilize octanoyl-CoA, raising the possibility that the substrate of the ligase reaction is octanoic acid itself. Although Lpl has been considered as an enzyme involved in lipoate salvage, the results imply that in T. kodakarensis, the TK1908 and TK1234 proteins function in de novo lipoyl-protein biosynthesis. IMPORTANCE Based on previous studies in bacteria and eukaryotes, lipoate-protein ligases (Lpls) have been considered to be involved exclusively in lipoate salvage. The genetic analyses in this study on the lipoate-protein ligase in T. kodakarensis, however, suggest otherwise and that the enzyme is additionally involved in de novo protein lipoylation. We also provide biochemical evidence that the lipoate-protein ligase displays broad substrate specificity and is capable of ligating acyl groups of various chain-lengths to the peptide substrate. We show that this apparent ambiguity in Lpl is resolved by the strict substrate specificity of the lipoyl synthase LipS in this organism, which only recognizes octanoyl-peptide. The results provide relevant physiological insight into archaeal protein lipoylation.

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Amino acid sequence analysis of the lipoyl and peripheral subunit-binding domains in the lipoate acetyltransferase component of the pyruvate dehydrogenase complex from Bacillus stearothermophilus.

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