Be condensed with the primer malonyl moiety retaining the carboxyl group introduced by acetyl-CoA carboxylase fixation of CO2. The other two malonyl-CoA molecules would lose their free carboxyl R1503 supplier groups in the course of the two decarboxylative Claisen reactions required to give the C7 dicarboxylate. This scheme is consistent with the 13C labeling studies and is chemically reasonable because type III polyketide synthases are known that use such a malonyl-primed mechanism to make dicarboxylic acids of odd carbon lengths in which one the two carboxyl groups is in thioester linkage (26, 27). However, in fatty acid synthesis the growing chains are attached to ACP rather than CoA and unlike polyketides, where the keto groups are retained or consumed in subsequent rearrangements of the carbon chain (e.g., cyclization), pimelate synthesis requires that the keto groups be converted to methylenes. Although the condensation, reduction and dehydration enzymes of fatty acid synthesis could perform the net reduction of the keto groups to methylenes required for assembly of the pimeloyl moiety, it seemed most unlikely that the fatty acid synthetic enzymes would be active on substrates having a carboxyl group in place of the usual terminal methyl group because the fatty acid synthetic enzymes sequester the growing fatty acyl chains in tunnels or clefts that are strongly hydrophobic (28). Recently it has been shown that this conundrum is avoided by “disguising” the terminal carboxyl group such that it can be recognized by the fatty acid synthesis enzymes (Fig. 2). Introduction of the disguise is the role of BioC which converts the free carboxyl group to its methyl ester by transfer of a methyl group from SAM. Methylation cancels the carboxyl group charge and provides a methyl carbon that mimics the methyl of the normal acyl chains. This methylated species has properties (chain length, hydrophobicity) approximating those of the substrates normally accepted by the enzymes of fatty acid synthesis. Following completion of the pimelic acid moiety the methyl ester would then be cleaved by BioH to give pimeloyl-ACP. This in turn would react with L-alanine in the BioF reaction to give 7-keto-8-aminopelargonic acid (KAPA), the first intermediate in assembly of the biotin ring structures (Fig. 2). BioH thus acts to free the carboxyl group that will eventually be used to attach biotin to the metabolic enzymes where it performs its key metabolic roles (29).Author Manuscript Author Manuscript Author Manuscript Author ManuscriptEcoSal Plus. Author manuscript; available in PMC 2015 January 06.CronanPageBioCAuthor Manuscript Author Manuscript Author Manuscript Author ManuscriptBioHPrior to the recent work nothing was known of the function of BioC, a protein of 28.3 kDa. It is highly conserved among the proteobacteria and is often annotated as a SAM-dependent methyl 11-Deoxojervine chemical information transferase. It had been proposed that BioC acts as a carrier protein that carries an intermediate transferred by BioH (30), but recent work disproves this notion. The BioC protein had not been studied biochemically probably because it invariably forms inclusion bodies upon overexpression (31). This recalcitrant property of BioC has precluded its direct analysis, although some activity was obtained upon denaturing and refolding the protein (13). The BioCs of close relatives of E. coli were as intractable as E. coli BioC and thus the BioCs of more diverse bacteria were tested. Expression of the BioC of Bacillus.Be condensed with the primer malonyl moiety retaining the carboxyl group introduced by acetyl-CoA carboxylase fixation of CO2. The other two malonyl-CoA molecules would lose their free carboxyl groups in the course of the two decarboxylative Claisen reactions required to give the C7 dicarboxylate. This scheme is consistent with the 13C labeling studies and is chemically reasonable because type III polyketide synthases are known that use such a malonyl-primed mechanism to make dicarboxylic acids of odd carbon lengths in which one the two carboxyl groups is in thioester linkage (26, 27). However, in fatty acid synthesis the growing chains are attached to ACP rather than CoA and unlike polyketides, where the keto groups are retained or consumed in subsequent rearrangements of the carbon chain (e.g., cyclization), pimelate synthesis requires that the keto groups be converted to methylenes. Although the condensation, reduction and dehydration enzymes of fatty acid synthesis could perform the net reduction of the keto groups to methylenes required for assembly of the pimeloyl moiety, it seemed most unlikely that the fatty acid synthetic enzymes would be active on substrates having a carboxyl group in place of the usual terminal methyl group because the fatty acid synthetic enzymes sequester the growing fatty acyl chains in tunnels or clefts that are strongly hydrophobic (28). Recently it has been shown that this conundrum is avoided by “disguising” the terminal carboxyl group such that it can be recognized by the fatty acid synthesis enzymes (Fig. 2). Introduction of the disguise is the role of BioC which converts the free carboxyl group to its methyl ester by transfer of a methyl group from SAM. Methylation cancels the carboxyl group charge and provides a methyl carbon that mimics the methyl of the normal acyl chains. This methylated species has properties (chain length, hydrophobicity) approximating those of the substrates normally accepted by the enzymes of fatty acid synthesis. Following completion of the pimelic acid moiety the methyl ester would then be cleaved by BioH to give pimeloyl-ACP. This in turn would react with L-alanine in the BioF reaction to give 7-keto-8-aminopelargonic acid (KAPA), the first intermediate in assembly of the biotin ring structures (Fig. 2). BioH thus acts to free the carboxyl group that will eventually be used to attach biotin to the metabolic enzymes where it performs its key metabolic roles (29).Author Manuscript Author Manuscript Author Manuscript Author ManuscriptEcoSal Plus. Author manuscript; available in PMC 2015 January 06.CronanPageBioCAuthor Manuscript Author Manuscript Author Manuscript Author ManuscriptBioHPrior to the recent work nothing was known of the function of BioC, a protein of 28.3 kDa. It is highly conserved among the proteobacteria and is often annotated as a SAM-dependent methyl transferase. It had been proposed that BioC acts as a carrier protein that carries an intermediate transferred by BioH (30), but recent work disproves this notion. The BioC protein had not been studied biochemically probably because it invariably forms inclusion bodies upon overexpression (31). This recalcitrant property of BioC has precluded its direct analysis, although some activity was obtained upon denaturing and refolding the protein (13). The BioCs of close relatives of E. coli were as intractable as E. coli BioC and thus the BioCs of more diverse bacteria were tested. Expression of the BioC of Bacillus.
