BioWF: A Naturally‐Fused, Di‐Domain Biocatalyst from Biotin Biosynthesis Displays an Unexpectedly Broad Substrate Scope

The carbon backbone of biotin is constructed from the C(7) di‐acid pimelate, which is converted to an acyl‐CoA thioester by an ATP‐dependent, pimeloyl‐CoA synthetase (PCAS, encoded by BioW). The acyl‐thioester is condensed with ʟ‐alanine in a decarboxylative, Claisen‐like reaction to form an aminoketone (8‐amino‐7‐oxononanoic acid, AON). This step is catalysed by the pyridoxal 5’‐phosphate (PLP)‐dependent enzyme (AON synthase, AONS, encoded by BioF). Distinct versions of Bacillus subtilis BioW (BsBioW) and E. coli BioF (EcBioF) display strict substrate specificity. In contrast, a BioW‐BioF fusion from Corynebacterium amycolatum (CaBioWF) accepts a wider range of mono‐ and di‐fatty acids. Analysis of the active site of the BsBioW : pimeloyl‐adenylate complex suggested a key role for a Phe (F192) residue in the CaBioW domain; a F192Y mutant restored the substrate specificity to pimelate. This surprising substrate flexibility also extends to the CaBioF domain, which accepts ʟ‐alanine, ʟ‐serine and glycine. Structural models of the CaBioWF fusion provide insight into how both domains interact with each other and suggest the presence of an intra‐domain tunnel. The CaBioWF fusion catalyses conversion of various fatty acids and amino acids to a range of AON derivatives. Such unexpected, natural broad substrate scope suggests that the CaBioWF fusion is a versatile biocatalyst that can be used to prepare a number of aminoketone analogues.