Pimeloyl-ACP methyl ester synthesis proceeds either artificially by acylation of ACP with the monomethyl ester of pimelate or physiologically by elongation of a primer molecule of malonyl-CoA methylated at its ω-carboxyl group by BioC. In the physiological pathway the committed step is the BioC-catalyzed SAM-dependent methylation of malonyl-CoA (or malonyl-ACP) . Malonyl-CoA methyl ester enters the fatty acid synthetic pathway as a dedicated biotin precursor and acts as primer (in place of the acetyl-CoA normally used in fatty acid synthesis) for the FabH catalyzed condensation reaction. The product of the proposed FabH reaction would be 3-ketoglutaryl-ACP methyl ester, a five-carbon dicarboxylate carrying the methyl ester moiety that allows the substrate to interact with the other fatty acid synthetic enzymes. 3-Ketoglutaryl-ACP methyl ester would be reduced by 3-ketoacyl-ACP reductase (FabG) to 3-hydroxylglutaryl-ACP methyl ester which would be dehydrated by 3-hydroxyacyl-ACP dehydratase (FabZ) to the enoyl-ACP methyl ester species. The double bond would be reduced by enoyl-ACP reductase (FabI) to give glutaryl-ACP methyl ester. The product of the first elongation cycle would then be elongated by a second decarboxylative Claisen condensation with malonyl-ACP (probably by FabB or FabF) to give 3-ketopimeloyl-ACP methyl ester which would be reduced and dehydrated as above to give pimeloyl-ACP methyl ester. BioH cleavage of the ester prevents further elongation and the resulting pimeloyl-ACP utilized by BioF to make KAPA which would begin synthesis of biotin. Abbreviations: DAPA, 7,8-diaminopelargonic acid; DTB, dethiobiotin; AMTOD, S-adenosyl-2-oxo-4-methylthiobutyric acid; SAH, S-adenosylhomocysteine; 5’-DOA, 5’-deoxyadenosine.

The cell free extract was prepared from the ΔbioC strain STL96 which carries multiple copies of all the bio operon genes except bioC. Purified and refolded BioC was added to all reactions except as shown. Bioassays were done using strain ER90 (ΔbioF) as described in Methods. (a) Standard DTB was bioassayed and reliably detected in levels as low as 1 pmol. (b) DTB synthesis specifically required malonyl-CoA as substrate. Structurally related compounds such as acetyl-CoA, succinyl-CoA and malonate were inactive. (c) DTB synthesis required malonyl-CoA, BioC, NADPH and all other reactions components as given for in vitro DTB synthesis in Methods. When pimeloyl-ACP methyl ester was added, both BioC and malonyl-CoA were omitted.

(a) KAPA synthesis in an extract lacking BioA with malonyl-CoA as substrate. The product was detected by bioassay on the ΔbioF strain STL115. When pimeloyl-ACP methyl ester was added both SAM and malonyl-CoA were omitted. (b) Requirement for the methyl ester moiety in conversion of glutaryl-ACP to DTB. The extract lacked BioC and the bioassay was performed on strain ER90 (ΔbioF bioC bioD). Glutaryl-ACP methyl ester was added to the reactions assayed in the two left hand quadrants whereas the reactions of the two right hand quadrants received glutaryl-ACP. (c) The requirement for active BioH in conversion of pimeloyl-ACP methyl ester to DTB. The extract lacked BioH with pimeloyl-ACP methyl ester as the substrate and the bioassay was performed using strain ER90. Either purified wild type BioH or the mutant BioH S82A protein (which lacks the active site nucleophile) was added to 20 µg/ml. The reaction of the lower right quadrant lacked extract, but contained both the wild type and mutant BioH proteins.

The microbiocide, triclosan, or the antibiotics cerulenin, thiolactomycin and sinefungin were added to the in vitro DTB synthesis with either malonyl-CoA (○) or pimeloyl-ACP methyl ester (■) as substrate. The level of DTB produced was estimated based on the area of the bioassay red zone and was standardized to the control reactions containing no inhibitor (100 % DTB synthesis was approximately 30 ρmol). The lack of inhibition seen when pimeloyl-ACP methyl ester was the substrate demonstrated that the amounts of inhibitor transferred to the assay disks were insufficient to inhibit growth of the bioassay strain ER90 (ΔbioF bioC bioD). Inhibition by triclosan was reduced when FabV, a triclosan resistant enoyl-ACP reductase, was added to 50 µg/ml in the reaction (●). The protein synthesis inhibitor, kanamycin, was tested with malonyl-CoA as the substrate and gave no inhibition over the same range of concentrations. A bioassay plate showing the effects of thiolactomycin on DTB synthesis is given at the lower right of the figure. Type of file: figure