Proved to be the case (215, 216). The fact that the lipoate of one of the T. acidophilum LplA structures was converted to lipoyl-AMP and that the locations of lipoate moieties of the two T. acidophilum LplA structures agreed well argues that these represent the catalytically competent lipoate binding site. It therefore follows that in the first E. coli LplA structure the lipoate molecule was bound in a catalytically inappropriate manner. Indeed, in a later report from the same group cocrystals of LplA with lipoyl-AMP and LplA with octyl-5-AMP and the apo form of GcvH. These structures define the LplA tructural mechanism. Three large scale conformational changes occur upon completion of the lipoate adenylation get MK-886 reaction i) the adenylate-binding, ii) theAuthor Manuscript Author Manuscript Author Manuscript Author ManuscriptEcoSal Plus. Author manuscript; available in PMC 2015 January 06.CronanPagelipoate-binding loops move to maintain the lipoyl-5-AMP reaction intermediate and iii) the C-terminal domain rotates by about 180 degrees. These changes are prerequisites for LplA to accommodate the apoprotein for the lipoate transfer reaction. The invariant Lys133 residue plays essential roles in both lthe ipoate adenylation and transfer steps. 3.2 Octanoyl-ACP:protein N-octanoyltransferase (LipB) During the characterization of E. coli lplA null mutant strains compelling evidence was found for a second protein lipoylation pathway that did not require the lplA gene product. When independently derived lplA null alleles were transduced into wild-type strains, the resulting mutant strains showed no growth defects on minimal glucose medium indicating that these strains possessed functional (therefore lipoylated) 2-oxoacid dehydrogenases. This was directly confirmed by bioassays that showed lplA null mutants to contain normal levels of lipoylated proteins (198). Thus, it was clear that E. coli has an lplA-independent lipoylation pathway. This was first attributed to a second ligase that had somehow been missed in the biochemical analyses perhaps due to the in vitro conditions chosen. However, no such second ligase could be found (206) and thus alternative pathways were considered. The most straightforward alternative pathway was that the fatty acid synthesis intermediate, octanoyl-ACP would be converted either directly or indirectly to lipoylated proteins. That is, lipoate synthesis would occur without a free carboxyl group. The carboxyl group would be bound in the thioester bond that links fatty acids to ACP and this bond would then be attacked by the -amino group of the lipoyl domain lysine residue to give the amide linkage. Several lines of evidence demonstrated that this alternative protein lipoylation pathway was dependent on the lipB gene product. The lipB gene was originally isolated as a class of lipoic acid auxotrophs (6). These mutants showed residual (leaky) growth in the absence of lipoic acid despite having putative null mutations due to transposon insertions into lipB (6, 217). This leakiness was reflected in their 2-oxo acid dehydrogenase activities and lipoylated protein contents. These strains retain about 20 of the enzyme activities and about 10 of lipoyl protein content of wild type strains (217). The leaky growth of lipB strains in the absence of lipoate was eliminated by introduction of an lplA mutation suggesting that lipB was Velpatasvir site involved in lipoyl domain modification as well as lipoate biosynthesis (198). Indeed, bioassays demonst.Proved to be the case (215, 216). The fact that the lipoate of one of the T. acidophilum LplA structures was converted to lipoyl-AMP and that the locations of lipoate moieties of the two T. acidophilum LplA structures agreed well argues that these represent the catalytically competent lipoate binding site. It therefore follows that in the first E. coli LplA structure the lipoate molecule was bound in a catalytically inappropriate manner. Indeed, in a later report from the same group cocrystals of LplA with lipoyl-AMP and LplA with octyl-5-AMP and the apo form of GcvH. These structures define the LplA tructural mechanism. Three large scale conformational changes occur upon completion of the lipoate adenylation reaction i) the adenylate-binding, ii) theAuthor Manuscript Author Manuscript Author Manuscript Author ManuscriptEcoSal Plus. Author manuscript; available in PMC 2015 January 06.CronanPagelipoate-binding loops move to maintain the lipoyl-5-AMP reaction intermediate and iii) the C-terminal domain rotates by about 180 degrees. These changes are prerequisites for LplA to accommodate the apoprotein for the lipoate transfer reaction. The invariant Lys133 residue plays essential roles in both lthe ipoate adenylation and transfer steps. 3.2 Octanoyl-ACP:protein N-octanoyltransferase (LipB) During the characterization of E. coli lplA null mutant strains compelling evidence was found for a second protein lipoylation pathway that did not require the lplA gene product. When independently derived lplA null alleles were transduced into wild-type strains, the resulting mutant strains showed no growth defects on minimal glucose medium indicating that these strains possessed functional (therefore lipoylated) 2-oxoacid dehydrogenases. This was directly confirmed by bioassays that showed lplA null mutants to contain normal levels of lipoylated proteins (198). Thus, it was clear that E. coli has an lplA-independent lipoylation pathway. This was first attributed to a second ligase that had somehow been missed in the biochemical analyses perhaps due to the in vitro conditions chosen. However, no such second ligase could be found (206) and thus alternative pathways were considered. The most straightforward alternative pathway was that the fatty acid synthesis intermediate, octanoyl-ACP would be converted either directly or indirectly to lipoylated proteins. That is, lipoate synthesis would occur without a free carboxyl group. The carboxyl group would be bound in the thioester bond that links fatty acids to ACP and this bond would then be attacked by the -amino group of the lipoyl domain lysine residue to give the amide linkage. Several lines of evidence demonstrated that this alternative protein lipoylation pathway was dependent on the lipB gene product. The lipB gene was originally isolated as a class of lipoic acid auxotrophs (6). These mutants showed residual (leaky) growth in the absence of lipoic acid despite having putative null mutations due to transposon insertions into lipB (6, 217). This leakiness was reflected in their 2-oxo acid dehydrogenase activities and lipoylated protein contents. These strains retain about 20 of the enzyme activities and about 10 of lipoyl protein content of wild type strains (217). The leaky growth of lipB strains in the absence of lipoate was eliminated by introduction of an lplA mutation suggesting that lipB was involved in lipoyl domain modification as well as lipoate biosynthesis (198). Indeed, bioassays demonst.
