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Journal of Bacteriology, March 2003, p. 1582-1589, Vol. 185, No. 5
0021-9193/03/$08.00+0     DOI: 10.1128/JB.185.5.1582-1589.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

The Escherichia coli lipB Gene Encodes Lipoyl (Octanoyl)-Acyl Carrier Protein:Protein Transferase

Sean W. Jordan1 and John E. Cronan, Jr.1,2*

Departments of Microbiology,1 Biochemistry, University of Illinois, Urbana, Illinois 618012

Received 10 October 2002/ Accepted 3 December 2002

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.

* Corresponding author. Mailing address: Department of Microbiology, University of Illinois, B103, Chemical and Life Sciences Laboratory, 601 S. Goodwin Ave., Urbana, IL 61801. Phone: (217) 333-7919. Fax: (217) 244-6697. E-mail: j-cronan{at}life.uiuc.edu.

Journal of Bacteriology, March 2003, p. 1582-1589, Vol. 185, No. 5
0021-9193/03/$08.00+0     DOI: 10.1128/JB.185.5.1582-1589.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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