Divergent evolution of an atypical S -adenosyl- l -methionine–dependent monooxygenase involved in anthracycline biosynthesis

Abstract

Bacterial secondary metabolic pathways are responsible for the biosynthesis of thousands of bioactive natural products. Many enzymes residing in these pathways have evolved to catalyze unusual chemical transformations, which is facilitated by an evolutionary pressure promoting chemical diversity. Such divergent enzyme evolution has been observed in S-adenosyl-l-methionine (SAM)-dependent methyltransferases involved in the biosynthesis of anthracycline anticancer antibiotics; whereas DnrK from the daunorubicin pathway is a canonical 4-O-methyltransferase, the closely related RdmB (52% sequence identity) from the rhodomycin pathways is an atypical 10-hydroxylase that requires SAM, a thiol reducing agent, and molecular oxygen for activity. Here, we have used extensive chimeragenesis to gain insight into the functional differentiation of RdmB and show that insertion of a single serine residue to DnrK is sufficient for introduction of the monooxygenation activity. The crystal structure of DnrK-Ser in complex with aclacinomycin T and S-adenosyl-l-homocysteine refined to 1.9-Å resolution revealed that the inserted serine S297 resides in an α-helical segment adjacent to the substrate, but in a manner where the side chain points away from the active site. Further experimental work indicated that the shift in activity is mediated by rotation of a preceding phenylalanine F296 toward the active site, which blocks a channel to the surface of the protein that is present in native DnrK. The channel is also closed in RdmB and may be important for monooxygenation in a solvent-free environment. Finally, we postulate that the hydroxylation ability of RdmB originates from a previously undetected 10-decarboxylation activity of DnrK.

A pharmaceutical model for the molecular evolution of microbial natural products

“…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 . In turn, this latter promiscuous activity has evolved to 10‐hydroxylation functionality as observed in RdmB from the rhodomycin pathway .…”

“…Natural product biosynthetic enzymes are ‘generalists’, which typically display slow reaction rates and substrate promiscuity . These enzymes have been widely shown to harbor weak secondary ‘moonlighting’ activities . This catalytic promiscuity promotes the ability to catalyze fortuitous chemical transformations and may form the seed for the emergence of new enzymatic activities .…”

“…This catalytic promiscuity promotes the ability to catalyze fortuitous chemical transformations and may form the seed for the emergence of new enzymatic activities . Furthermore, natural product biosynthetic pathways often encode multifunctional enzymes that catalyze either several consecutive reactions with different regiospecificity or entirely different chemical transformations .…”

“…This is most apparent in comparisons of related secondary metabolite biosynthetic gene clusters, where highly conserved enzymes can nonetheless catalyze distinct chemistry. Perhaps the most illustrative examples are found from evolution‐inspired protein engineering experiments, where the activities of functionally distinct enzymes can be interchanged with single point mutations .…”

“…In turn, this latter promiscuous activity has evolved to 10‐hydroxylation functionality as observed in RdmB from the rhodomycin pathway . The shift in catalysis could be pinpointed to insertion of a single serine residue S297 to DnrK, which resulted in gain of 10‐hydroxylation activity . Surprisingly, the inserted serine points away from the active site in DnrK‐Ser, but since it is located in α16‐helix, the insertion results in rotation of the preceding phenylalanine F296 toward the active site.…”