Lipoate-protein and biotin-protein ligases of Escherichia coli
Morris, Timothy William
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Permalink
https://hdl.handle.net/2142/20505
Description
Title
Lipoate-protein and biotin-protein ligases of Escherichia coli
Author(s)
Morris, Timothy William
Issue Date
1994
Doctoral Committee Chair(s)
Cronan, John E.
Department of Study
Microbiology
Discipline
Microbiology
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Biology, Genetics
Biology, Microbiology
Chemistry, Biochemistry
Language
eng
Abstract
Lipoate and biotin are critical and almost universally distributed cofactors which share striking parallels. Both cofactors are covalently attached to specific lysine residues of specific apoprotein domains, and thus function as protein bound carriers of reaction intermediates between separate active sites of multienzyme complexes. Escherichia coli synthesizes both compounds and also expresses a set of dedicated, highly specific cofactor-apoprotein ligases to generate protein bound lipoyl and biotinyl groups.
I have isolated and characterized an E. coli gene (lplA) which encodes the first known lipoate-protein ligase. Strains with lplA null mutations displayed severe defects in the incorporation of exogenously supplied lipoate and lipoate analogs, were highly resistant to selenolipoate (a selenium containing lipoate analog), and contained no detectable lipoate-protein ligase activity in cell extracts. The lplA gene was cloned, sequenced, and used to overexpress the 38 kDa LplA protein. Purified LplA catalyzed the ATP dependent attachment of lipoate to apoprotein. The previously identified slr1 selenolipoate resistance mutation (Reed et al., 1994 PNAS 91:3720) was redesignated lplA1 after sequencing revealed that this mutation encoded a G76S substitution in the LplA lipoate ligase.
Analysis of lplA null mutants revealed the existence of a second lipoyl ligase enzyme as well as an unexpected redundancy between the functions of lplA and the previously isolated lipB gene (Reed and Cronan, 1993 J. Bacteriol. 175:1325). I found that lplA null mutants displayed no growth defects unless combined with lipA (lipoate synthesis) or lipB mutations, and that overexpression of wild type lplA suppressed lipB null mutations. Assays of growth, transport, lipoyl-protein content, and immunoblotting experiments demonstrated that the lplA gene encoded a ligase for the incorporation of exogenously supplied lipoate whereas the lipB gene was required for function of the second lipoyl ligase, which utilized only the lipoyl groups generated via endogenous (lipA mediated) biosynthesis. This lipB dependent ligase was further shown to cause the accumulation of aberrantly modified octanoyl-proteins in lipoate deficient cells. These results were used to construct a two pathway/two ligase model of lipoate metabolism in E. coli.
A recombinant fusion protein system was also developed to study structure-function relationships in the biotin accepting domain of the BCCP subunit of acetyl-CoA carboxylase. This analysis suggested that an intact folded apodomain plus a flexible linker sequence were required for efficient biotinylation by the biotin ligase. Localized mutagenesis plus selection for biotinylation defective mutants identified three regions of the BCCP biotin domain as potential determinants of apoprotein/ligase interactions.
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