LAUSR

Macrolactins (MLNs) are a class of important antimacular degeneration and antitumor agents. Malonylated/succinylated MLNs are even more important due to their efficacy to overcome multidrug resistant bacteria. However, which enzyme catalyzes this reaction remains enigmatic. Herein, we deciphered a beta-lactamase homolog BmmI to be responsible for this step. BmmI could specifically attach C3-C5 alkyl acid thioesters onto 7-OH of MLN A, and also exhibits substrate promiscuity towards acyl acceptors with different scaffolds. Crystal structure of BmmI covalently linked with succinyl group and systematic mutagenesis highlighted the role of an oxyanion hole-like geometry in the recognition of carboxyl-terminated acyl donors. Engineering of this geometry expanded its substrate scope, with the R166A/G/Q variants recognizing up to C12 alkyl acid thioester. Structure of BmmI with the acyl acceptor MLN A revealed the importance of Arg292 in recognition of the macrolide substrates. Moreover, the mechanism of BmmI-catalyzed acyltransfer reaction was established, unmasking the deft role of Lys76 in governing acyl donors as well as catalysis. Our studies uncover the delicate mechanism underlying substrate selectivity of acyltransferases, which would guide rational enzyme engineering for drug development.