Abstract
Recent work implicated the Escherichia coli BioC protein as the initiator of the synthetic pathway that forms the pimeloyl moiety of biotin (Lin, S., Hanson, R. E., and Cronan, J. E. (2010) Nat. Chem. Biol. 6, 682-688). BioC was believed to be an O-methyltransferase that methylated the free carboxyl of either malonyl-CoA or malonyl-acyl carrier protein based on the ability of O-methylated (but not unmethylated) precursors to bypass the BioC requirement for biotin synthesis both in vivo and in vitro. However, only indirect proof of the hypothesized enzymatic activity was obtained because the activities of the available BioC preparations were too low for direct enzymatic assay. Because E. coli BioC protein was extremely recalcitrant to purification in an active form, BioC homologues of other bacteria were tested. We report that the native form of Bacillus cereus ATCC10987 BioC functionally replaced E. coli BioC in vivo, and the protein could be expressed in soluble form and purified to homogeneity. In disagreement with prior scenarios that favored malonyl-CoA as the methyl acceptor, malonyl-acyl carrier protein was a far better acceptor of methyl groups from S-adenosyl-L-methionine than was malonyl-CoA. BioC was specific for the malonyl moiety and was inhibited by S-adenosyl-L-homocysteine and sinefungin. High level expression of B. cereus BioC in E. coli blocked cell growth and fatty acid synthesis.
Highlights
The pimelate moiety of biotin is made by a modified fatty acid synthesis pathway
We report that the native form of Bacillus cereus ATCC10987 BioC functionally replaced E. coli BioC in vivo, and the protein could be expressed in soluble form and purified to homogeneity
Soluble Expression and Purification of B. cereus BioC—A large assortment of expression constructs, growth conditions, and host cells failed to give soluble E. coli BioC and we resorted to screening putative BioCs from other bacteria
Summary
The pimelate moiety of biotin is made by a modified fatty acid synthesis pathway. Results: The first reaction is O-methylation of the free carboxyl of malonyl-acyl carrier protein. BioC was believed to be an O-methyltransferase that methylated the free carboxyl of either malonyl-CoA or malonyl-acyl carrier protein based on the ability of O-methylated (but not unmethylated) precursors to bypass the BioC requirement for biotin synthesis both in vivo and in vitro. BioC catalyzes transfer of the methyl group of S-adenosyl-L-methionine (SAM) to the -carboxyl group of malonyl-thioester of either CoA or acyl carrier protein (ACP) to form an O-methyl ester. Methylation of the free carboxyl group of the malonyl-thioester was essential because of the extremely hydrophobic nature of the active sites of the fatty acid synthesis proteins [9]. Two rounds of the standard fatty acid reductase-dehydratase-reductase reaction sequence results in the ACP thioester of -methyl pimelic acid (Fig. 1) At this stage the methyl group must be removed by BioH to block further elongation to azelayl-ACP methyl ester, a physiologically useless product [5]. We report the enzymatic properties of Bacillus cereus BioC
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