Investigation of the Iterative Methylations by a Cobalamin‐Dependent Radical SAM Methyltransferase

Abstract

Natural products with branched alkoxy groups have been used widely in the development of bioactive compounds. However, the biosynthesis of branched alkoxy groups like the unique t‐butyl group is still poorly understood. The cystobactamids are a novel class of isopropyl substituted antibacterial compounds produced by myxobacteria. In the biosynthesis gene cluster of cystobactamid, CysS is assigned as a cobalamin‐dependent radical S‐adenosylmethionine (SAM) enzyme, which is proposed to catalyze the iterative methylations on the 3‐methoxy‐4‐aminobenzoic acid moieties of cystobactamids. To reconstitute CysS, different substrates analogs were synthesized. When the pantetheinyl substrate analogs were used, multiple methylations were observed to form ethyl, isopropyl, t‐butyl and sec‐butyl substituted products from a substrate methyl ether by CysS. These cobalamin dependent methylations also produce 5′‐deoxyadenosine(5′dA) and S‐adenosylhomocysteine (SAH) in equal amounts. The isotope labeling experiments suggest that the methyl group from SAM is first transferred to cobalamin followed by the attack of the substrate radical to give methylated products. A combinatorial antibiotic library of 25 cystobactamid analogs could potentially be built using CysS by Cystobacter sp. Different substituted substrate analogs were designed to trap the organic radical species, cobalamin intermediates and probe the radical substitution step. Here, we have showed the first in vitro reconstitution of a cobalamin‐dependent radical SAM enzyme catalyzing the conversion of a methyl group to a t‐butyl group by sequential methylations. The detailed mechanistic studies help us to unravel how CysS uses SAM, [4Fe–4S] cluster and cobalamin to perform iterative methylations on sp3‐hybridized carbon centers and better understand the mechanism of other widespread cobalamin‐dependent radical SAM methyltransferases.Support or Funding InformationThis work was supported by NIH (DK44083) and by the Robert A. Welch Foundation (A‐0034).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.