A set of closely related methyltransferases for site-specific tailoring of anthraquinone pigments

Modification of the polyketide anthraquinone AQ-256 in the entomopathogenic Photorhabdus luminescens involves several O-methylations, but the biosynthetic gene cluster antA-I lacks corresponding tailoring en-zymes. We here describe the identification of five putative, highly homologous O-methyltransferases en-coded in the genome of P. luminescens. Activity assays in vitro and deletion experiments in vivo revealed that three of them account for anthraquinone tailoring by producing three monomethylated and two dimethy-lated species of AQ-256. X-ray structures of all five enzymes indicate high structural and mechanistic simi-larity. As confirmed by structure-based mutagenesis, a conserved histidine at the active site likely functions as a general base for substrate deprotonation and subsequent methyl transfer in all enzymes. Eight complex structures with AQ-256 as well as mono-and dimethylated derivatives confirm the substrate specificity pat-terns found in vitro and visualize how single amino acid differences in the active-site pockets impact sub-strate orientation and govern site-specific methylation.