Mechanistic studies on CysS - a Vitamin B12-dependent radical SAM methyltransferase involved in the biosynthesis of the t-butyl group of cystobactamid.

Cobalamin (Cbl)-dependent radical SAM methyltransferases catalyze methylation reactions at non-nucleophilic centers in a wide range of substrates. CysS is a Cbl-dependent radical SAM methyltransferase involved in cystobactamid biosynthesis. This enzyme catalyzes the sequential methylation of a methoxy group to form ethoxy, i-propoxy, s-butoxy, and t-butoxy groups on a p-aminobenzoate peptidyl carrier protein thioester intermediate. This biosynthetic strategy enables the host myxobacterium to biosynthesize a combinatorial antibiotic library of 25 cystobactamid analogs. In this paper, we describe three experiments to elucidate how CysS uses Cbl, SAM, and a [4Fe- 4S] cluster to catalyze iterative methylation reactions: a cyclopropylcarbinyl rearrangement was used to trap the substrate radical and to estimate the rate of the radical substitution reaction involved in the methyl transfer; a bromoethoxy analog was used to explore the active site topography; and deuterium isotope effects on the hydrogen atom abstraction by the adenosyl radical were used to investigate the kinetic significance of the hydrogen atom abstraction. Based on these experiments, a revised mechanism for CysS is proposed.

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