Fig. 1: Dual site-selective crosslinking of the mycocerosic acid synthase (MAS).

a Depiction of ACP processivity in MAS biosynthesis. ACP must interact with each catalytic domain, acyltransferase (AT, step 5 extension unit loading), ketosynthase (KS, step 1 condensation. Substrate loading or reloading step is not shown), ketoreductase (KR, step 2 β-ketoreduction), dehydratase (DH, step 3 dehydration), and enoylreductase (ER, step 4 α,β-enoylreduction), through discrete protein-protein and protein-substrate interactions during each catalytic cycle. MAS can be divided into two compartments, one that serves to load and elongate acyl chains (condensation compartment, steps 1 and 5); and a second that serves to modify and fully reduce the condensation product (modification compartment, steps 2–4). MAS exists as a homodimer, where protomer 1 (indicated by ^A after each abbreviation) is depicted with domain abbreviations, and protomer 2 (indicated by ^B or by prime’ after each abbreviation). b Mechanism of ACP = KS (=indicates crosslink) crosslinking reaction. c Mechanism of ACP = DH crosslinking reaction. d This process is achieved through chemoenzymatic loading of 1 onto the apo-ACP to deliver crypto-ACP. Once loaded, the active site Cys of KS or His of DH react with the α-Br-pantetheinamide warhead, yielding a crosslinked complex.