Evolutionary Trajectories for the Functional Diversification of Anthracycline Methyltransferases

Abstract

Microbial natural products are an important source of chemical entities for drug discovery. Recent advances in understanding the biosynthesis of secondary metabolites has revealed how this rich chemical diversity is generated through functional differentiation of biosynthetic enzymes. For instance, investigations into anthracycline anticancer agents have uncovered distinct S-adenosyl methionine (SAM)-dependent proteins: DnrK is a 4-O-methyltransferase involved in daunorubicin biosynthesis, whereas RdmB (52% sequence identity) from the rhodomycin pathway catalyzes 10-hydroxylation. Here, we have mined unknown anthracycline gene clusters and discovered a third protein subclass catalyzing 10-decarboxylation. Subsequent isolation of komodoquinone B from two Streptomyces strains verified the biological relevance of the decarboxylation activity. Phylogenetic analysis inferred two independent routes for the conversion of methyltransferases into hydroxylases, with a two-step process involving loss-of-methylation and gain-of-hydroxylation presented here. Finally, we show that simultaneously with the functional differentiation, the evolutionary process has led to alterations in substrate specificities.

A pharmaceutical model for the molecular evolution of microbial natural products

“…However, Ohno's law of Evolution by Gene Duplication would appear not to apply to secondary metabolism. A growing body of evidence suggests that natural product gene functions may also evolve directly in the biosynthetic gene clusters without gene duplication , which is possible because genes involved in secondary metabolism are not essential for the survival of the host. This is most apparent in comparisons of related secondary metabolite biosynthetic gene clusters, where highly conserved enzymes can nonetheless catalyze distinct chemistry.…”

“…These promiscuous enzymes have diversified to catalyze 4‐O‐methylation, 10‐decarboxylation, or 10‐hydroxylation, with additional changes in substrate specificity in regard to the glycosylation state . The functional diversification appears to have occurred directly in situ within the gene clusters (Fig B), as demonstrated by the congruence of phylogenetic trees composed of the methyltransferases and core anthracycline biosynthetic genes . The canonical member DnrK from the daunorubicin pathway is a 4‐O‐methyltransferase , but it harbors moonlighting 10‐decarboxylation activity toward anthracyclines with a free carboxyl group at C10 .…”

“…A). Even though the structural basis for 10‐decarboxylation is still unresolved, the loss of methylation activity could result from misalignment of the substrate in relation to SAM, which would leave 10‐decarboxylation as the sole functionality for EamK . Interestingly, 10‐hydroxylation may offer the producing organism significant selective advantage, since this functionality appears to have evolved independently on at least two occasions as exemplified by the CalMB‐type proteins (Fig.…”

Evolution-Inspired Engineering of Anthracycline Methyltransferases

“…Tailoring steps catalysed by SAM methyltransferase-like proteins 29 have yielded particular insight into evolution of anthracyclines and generation of structural diversity. DnrK is a canonical S -adenosyl-L-methionine (SAM) -dependent methyltransferase that catalyses 4-O-methylation (Fig.…”

“…However, the activity depends on the presence of a free 10-carboxyl group in the substrate generated by aclacinomycin 15-methylesterases such as EamC and DnrP (Fig. 1b) 29 . DnrK is quite specific with respect to the length of the carbohydrate chain at C-7, accepting only monoglycosides (Fig.…”