“Lipoic Acid Biosynthesis in Bacillus subtilis 168: A novel Octanoyltransferase isoenzyme”

QUIN CHRISTENSEN; MARTIN, N.; MANSILLA, MC; DE MENDOZA, D; JOHN CRONAN

San Diego

Congreso; 110th General Meeting of the American Society for Microbiology.; 2010

ASM

Lipoic acid is a cofactor required for energy-conserved aerobic oxidation of 2-oxoacids and glycine.  The genes for lipoic acid metabolism were initially identified in Escherichia coli over ten years ago, but it is clear that many other variants exist.  An advantage of whole genome sequencing is that it allows determination of what genes are present, but also those that are absent.  It has been previously observed that Bacillus subtilis 168 lacks a homologue of E. coli octanoyl-[acyl carrier protein]:protein N-octanoyltransferase, LipB.  The gene encoding LipB is essential for lipoic acid biosynthesis in E. coli, yet B. subtilis 168 is a lipoic acid prototroph.  Through genetic complementation of an E. coli lipB mutant we have identified a complementing gene from B. subtilis 168, which we have named lipM.  Disruption of the gene in the host leads to lipoic acid auxotrophy.  We have demonstrated octanoyltransfer activity of LipM in vitro and in vivo using various lipoyl domain substrates.  By LC-MS-MS analysis we have identified the acyl-enzyme intermediate on a LipM cysteine residue analogous, but not homologous, to LipB.  From phylogenetic analysis we find LipM shares a common ancestor with lipoic acid ligases.  As such, it represents a new octanoyltransferase isozyme for lipoic acid biosynthesis.  Characterization of two other lipoic acid ligase homologues of B. subtilis has also been performed.  One, which we have named LplJ, is a canonical lipoic acid ligase.  The other, which we have named LipL, is required for lipoic acid biosynthesis but its role is the subject of further study.  B. subtilis lipoic acid metabolism has proven to be unexpectedly complicated and further analyses will lead to a more complete picture of lipoic acid metabolism.