Affiliation:
1. Departments of Microbiology
2. Biochemistry, University of Illinois, Urbana, Illinois 61801
Abstract
ABSTRACT
In an earlier study (S. W. Jordan and J. E. Cronan, Jr., J. Biol. Chem. 272:17903-17906, 1997) we reported a new enzyme, lipoyl-[acyl carrier protein]-protein
N
-lipoyltransferase, in
Escherichia coli
and mitochondria that transfers lipoic acid from lipoyl-acyl carrier protein to the lipoyl domains of pyruvate dehydrogenase. It was also shown that
E. coli lipB
mutants lack this enzyme activity, a finding consistent with
lipB
being the gene that encoded the lipoyltransferase. However, it remained possible that
lipB
encoded a positive regulator required for lipoyltransferase expression or action. We now report genetic and biochemical evidence demonstrating that
lipB
encodes the lipoyltransferase. A
lipB
temperature-sensitive mutant was shown to produce a thermolabile lipoyltransferase and a tagged version of the
lipB
-encoded protein was purified to homogeneity and shown to catalyze the transfer of either lipoic acid or octanoic acid from their acyl carrier protein thioesters to the lipoyl domain of pyruvate dehydrogenase. In the course of these experiments the ATG initiation codon commonly assigned to
lipB
genes in genomic databases was shown to produce a nonfunctional
E. coli
LipB protein, whereas initiation at an upstream TTG codon gave a stable and enzymatically active protein. Prior genetic results (T. W. Morris, K. E. Reed, and J. E. Cronan, Jr., J. Bacteriol. 177:1-10, 1995) suggested that lipoate protein ligase (LplA) could also utilize (albeit poorly) acyl carrier protein substrates in addition to its normal substrates lipoic acid plus ATP. We have detected a very slow LplA-catalyzed transfer of lipoic acid and octanoic acid from their acyl carrier protein thioesters to the lipoyl domain of pyruvate dehydrogenase. A nonhydrolyzable lipoyl-AMP analogue was found to competitively inhibit both ACP-dependent and ATP-dependent reactions of LplA, suggesting that the same active site catalyzes two chemically diverse reactions.
Publisher
American Society for Microbiology
Subject
Molecular Biology,Microbiology
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