The predominance of nucleotidyl activation in bacterial phosphonate biosynthesis.

Phosphonates are rare and unusually bioactive natural products. However, most bacterial phosphonate biosynthetic capacity is dedicated to tailoring cell surfaces with molecules like 2-aminoethylphosphonate (AEP). Although phosphoenolpyruvate mutase (Ppm)-catalyzed installation of C-P bonds is known, subsequent phosphonyl tailoring (Pnt) pathway steps remain enigmatic. Here we identify nucleotidyltransferases in over two-thirds of phosphonate biosynthetic gene clusters, including direct fusions to ~60% of Ppm enzymes. We characterize two putative phosphonyl tailoring cytidylyltransferases (PntCs) that prefer AEP over phosphocholine (P-Cho) - a similar substrate used by the related enzyme LicC, which is a virulence factor in Streptococcus pneumoniae. PntC structural analyses reveal steric discrimination against phosphocholine. These findings highlight nucleotidyl activation as a predominant chemical logic in phosphonate biosynthesis and set the stage for probing diverse phosphonyl tailoring pathways.

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

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.