PokMT1 from the Polyketomycin Biosynthetic Machinery of <i>Streptomyces diastatochromogenes</i> Tü6028 Belongs to the Emerging Family of <i>C</i>‑Methyltransferases That Act on CoA-Activated Aromatic Substrates

Recent biochemical characterizations of the MdpB2 CoA ligase and MdpB1 <i>C</i>-methyltransferase (<i>C</i>-MT) from the maduropeptin (MDP, <b>2</b>) 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, <b>3</b>). Biochemical characterization unambiguously established that PokM3 and PokMT1 catalyze the sequential conversion of 6-methylsalicylic acid (6-MSA, <b>4</b>) to form 3,6-dimethylsalicylyl-CoA (3,6-DMSA-CoA, <b>6</b>), which serves as the direct precursor for the 3,6-dimethylsalicylic acid (3,6-DMSA) moiety in the biosynthesis of <b>3</b>. PokMT1 catalyzes the C-methylation of 6-methylsalicylyl-CoA (6-MSA-CoA, <b>5</b>) with a <i>k</i>_cat of 1.9 min^–1 and a <i>K</i>_m of 2.2 ± 0.1 μM, representing the most proficient <i>C</i>-MT characterized to date. Bioinformatics analysis of MTs from natural product biosynthetic machineries demonstrated that PokMT1 and MdpB1 belong to a phylogenetic clade of <i>C</i>-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 <b>5</b>. 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 <i>C</i>-MTs in biocatalysis and synthetic biology