Fig. 3: A role of BioZ in the FAS II-involving biotin biosynthesis.

a In vitro biosynthesis of pimeloyl-ACP and/or its precursors. Using the FAS II system, BioZ catalyzes the synthesis reaction of pimeloyl-ACP from malonyl-ACP and glutaryl-CoA (ACP). The reaction mixture was separated with conformation-sensitive urea polyacrylamide gel electrophoresis (PAGE). A representative result is given from three trials. Of note: 17.5% PAGE (pH 9.5) containing 0.5 M urea was used here. The two controls (C4-ACP and C6-ACP) served as standards/markers for this conformation-sensitive urea gel. C7-ACP denotes four species of ACP attached with an acyl seven-carbon fatty acyl chain, namely, 5-keto-pimeloyl-ACP, 5-hydroxyl-pimeloyl-ACP, enoyl-pimeloyl-ACP, and pimeloyl-ACP. b MS/MS identification of 5-keto-pimeloyl-ACP, a primary product from the BioZ reaction coupled with FAS II, using glutaryl-CoA and malonyl-ACP as substrates. The use of MS/MS allowed us to detect the presence of four C7-ACP species in the above reaction system. As for an initial product of BioZ reaction, 5-keto-pimeloyl ACP, a 15-residue peptide fragment of interest is given. The C7-fatty acyl modification with high reliability is localized on the conserved Serine 36 of ACP. The two peaks of peptide fragments indicated with pink arrows were used to determine C7 acyl modification. The resultant mass was 498.1533, which is close to the theoretical mass (498.143) of Ppan-linked keto-pimeloyl moiety. Of note, it might be not as stable as pimelic acid. c Cartoon illustration of the pimeloyl-ACP structure. This was generated from the complex structure of methyl-pimeloyl-ACP and BioH (PDB: 4ETW) with appropriate modifications. d A scheme for BioZ bypassing the canonical early steps of biotin synthesis. Unlike the paradigm “BioC-BioH” mechanism of biotin synthesis (above the dashed line), the BioZ reaction bypasses the earlier steps of “BioC-BioH” in biotin biosynthesis (below the dashed line). Recently, glutaryl-CoA is determined to physiologically arise from lysine catabolism in Agrobacterium species⁴⁷. Designations: C4-ACP butanoyl-ACP, C6-ACP hexanoyl-ACP, Mal-ACP malonyl-ACP, Glu-ACP glutaryl-ACP, Pim-ACP pimeloyl-ACP.