Aclacinomycin 10-Hydroxylase Is a Novel Substrate-assisted Hydroxylase Requiring S-Adenosyl-l-methionine as Cofactor

Abstract

Aclacinomycin 10-hydroxylase is a methyltransferase homologue that catalyzes a S-adenosyl-L-methionine (AdoMet)-dependent hydroxylation of the C-10 carbon atom of 15-demethoxy-⑀-rhodomycin, a step in the biosynthesis of the polyketide antibiotic ␤-rhodomycin. SAdenosyl-L-homocysteine is an inhibitor of the enzyme, whereas the AdoMet analogue sinefungin can act as cofactor, indicating that a positive charge is required for catalysis.18 O 2 experiments show that the hydroxyl group is derived from molecular oxygen.

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

“…This loop region is disordered in the RdmB-SAM binary complex (25), whereas it is well defined in the ternary complex containing 11-deoxy-β-rhodomycin A (6; Fig. 1) (26). In addition, in comparison with DnrK (22), the loop region has moved closer to the ligand in RdmB, suggesting that R1 may be important for the 10-hydroxylation activity and/or the entry of the ligand into the active site.…”

“…Despite these changes, no 4-O-methylation activity was detected with this substrate in any of the chimeric enzymes, which would indicate that the aglycone is still not correctly positioned in terms of geometry in the active-site cavity. The lack of methylation activity in native RdmB has been suggested to occur due to wrong alignment of the substrate for a methyl transfer reaction using a S N 2 mechanism, where the methyl group has to be in line with the oxygen of the substrate as well as the sulfur atom of SAM for the reaction to proceed (26).…”

“…1), which has been noted as the likely explanation for why the enzyme only accepts monoglycosylated substrates (22). The corresponding region in RdmB is rather different and the carbohydrate chain extends from the binding pocket into the bulk solution in the RdmB ternary complex (26), which prompted us to select this region (R3; Fig. S1) as the third segment for chimeragenesis.…”

“…2) as substrates. The reaction mixtures also included SAM, which is essential as a cosubstrate for DnrK (22) and as a cofactor for RdmB (26), and a reducing agent (DTT) that is required solely by RdmB (26). The reaction with native DnrK resulted in a single main product ( Fig.…”

“…Determination of the crystal structure of RdmB confirmed that the enzyme binds SAM (25) and that the ternary complex is indeed very similar to that of DnrK with a root-meansquare deviation of 1.14 Å for 335 equivalent Cα atoms (26). Despite these features, RdmB completely lacks methyltransferase activity (24,27,28); it is an anthracycline 10-hydroxylase, which requires SAM, molecular oxygen, and a thiol reducing agent for activity (26).…”