LAUSR

Recent biochemical characterizations of the MdpB2 CoA ligase and MdpB1 C-methyltransferase (C-MT) from the maduropeptin (MDP, 2) biosynthetic machinery revealed unusual pathway logic involving C-methylation occurring on a CoA-activated aromatic substrate. Here we confirmed this pathway logic for the biosynthesis of polyketomycin (POK, 3). Biochemical characterization unambiguously established that PokM3 and PokMT1 catalyze the sequential conversion of 6-methylsalicylic acid (6-MSA, 4) to form 3,6-dimethylsalicylyl-CoA (3,6-DMSA-CoA, 6), which serves as the direct precursor for the 3,6-dimethylsalicylic acid (3,6-DMSA) moiety in the biosynthesis of 3. PokMT1 catalyzes the C-methylation of 6-methylsalicylyl-CoA (6-MSA-CoA, 5) with a kcat of 1.9 min-1 and a Km of 2.2 ± 0.1 μM, representing the most proficient C-MT characterized to date. Bioinformatics analysis of MTs from natural product biosynthetic machineries demonstrated that PokMT1 and MdpB1 belong to a phylogenetic clade of C-MTs that preferably act on aromatic acids. Significantly, this clade includes the structurally characterized enzyme SibL, which catalyzes C-methylation of 3-hydroxykynurenine in its free acid form, using two conserved tyrosine residues for catalysis. A homology model and site-directed mutagenesis suggested that PokMT1 also employs this unusual arrangement of tyrosine residues to coordinate C-methylation but revealed a large cavity capable of accommodating the CoA moiety tethered to 5. CoA activation of the aromatic acid substrate may represent a general strategy that could be exploited to improve catalytic efficiency. This study sets the stage to further investigate and exploit the catalytic utility of this emerging family of C-MTs in biocatalysis and synthetic biology.