Cooperative mechanisms of β-ketoacyl-ACP synthase III and acyl carrier protein in branched-chain fatty acid biosynthesis in Cutibacterium acnes.

The branched-chain fatty acids (BCFAs) synthesized by Cutibacterium acnes contribute to its membrane fluidity and adaptation to anaerobic environments. We investigated the structural and functional characteristics of β-ketoacyl-ACP synthase III (CaKAS III) and its cofactor, acyl carrier protein (CaACP) that are key components of BCFA synthesis. Structural analysis revealed that W228 and V233 in CaKAS III regulate branched-chain substrate accommodation. Docking and molecular dynamics (MD) simulations demonstrated that isovaleryl-CoA is stably accommodated in the CaKAS III cavity but does not readily enter the cavity of the W228V/V233L mutant or KAS III in E. coli, which does not produce BCFAs. These findings, consistent with the results of conformation-sensitive urea PAGE, highlight the critical roles of W228 and V233 in substrate selectivity. NMR titration identified key CaKAS III-CaACP interaction interface, with the α2 and α3-helices of CaACP acting as major surfaces for substrate transfer. Comparative MD simulations of CaACP and ACPs from BCFA-producing species Staphylococcus aureus and Bacillus subtilis, as well as Escherichia coli ACP (EcACP), indicated stable isoheptanoyl accommodation in CaACP and other ACPs, but not in the EcACP cavity. We propose that CaKAS III primarily dictates BCFA substrate specificity via its active-site architecture, while CaACP facilitates the accommodation and transfer of BCFA intermediates. These findings highlight the cooperative mechanisms governing substrate specificity and the roles of CaKAS III and CaACP in BCFA synthesis, presenting potential targets for novel antibacterial strategies.