Thioester Bond Formation Catalyzed by CalO4: A β-Ketoacyl-ACP Synthase III-like Acyltransferase in Calicheamicin Biosynthesis

β-Ketoacyl-ACP synthase (KAS) III enzymes play central roles in fatty acid biosynthesis and the production of various natural products. While their canonical function involves catalyzing C-C bond formation between acetyl-CoA and malonyl acyl-carrier-proteins (ACPs), an increasing number of non-canonical activities have been reported among KAS III homologues, including C-O and C-N bond formation. Here, we describe a KAS III family enzyme, CalO4, that catalyzes C-S bond formation during the biosynthesis of the potent antitumor agent calicheamicin. We successfully reconstituted its transacylation activities, demonstrated C-S bond formation, and assessed its substrate selectivity. Furthermore, X-ray crystallography combined with molecular docking and mutational analysis identified key residues likely involved in substrate selection and catalysis. This work not only expands the functional diversity of the KAS III family but also provides a potential biocatalyst for hindered C-S bond formation and a target for engineering novel calicheamicin-like or other bioactive compounds.