Accumulation of an unprecedented 5′-deoxyadenos-4′-yl radical unmasks the kinetics of the radical-mediated C-C bond formation step in MoaA catalysis

Abstract

AbstractRadicalS-adenosyl-L-methionine (SAM) enzymes catalyze various free radical-mediated reactions. In these enzymes, the rate-determining SAM cleavage kinetically masks all the subsequent steps. Due to this kinetic masking, detailed mechanistic characterization of radical transformations catalyzed by these enzymes is very difficult. Here, we report a successful kinetic characterization of the radical C-C bond formation catalyzed by a MoaA radical SAM enzyme. MoaA catalyzes an unprecedented 3′,8-cyclization of GTP into 3′,8-cyclo-7,8-dihydro-GTP (3′,8-cH2GTP) during the molybdenum cofactor (Moco) biosynthesis. Through a series of EPR and biochemical characterization, we found that MoaA accumulates a 5′-deoxyadenos-4′-yl radical (5′-dA-C4′•) under the turnover conditions, and forms (4′S)-5′-deoxyadenosine ((4′S)-5′-dA), which is a C-4′ epimer of the naturally occurring (4′R)-5′-dA. Together with kinetic characterizations, these observations revealed the presence of a shunt pathway in which an on-pathway intermediate, GTP C-3′ radical, abstracts H-4′ atom from 5′-dA to transiently generate 5′-dA-C4′• that is subsequently reduced stereospecifically to yield (4′S)-5′-dA. Detailed kinetic characterization of the shunt and the main pathways provided the comprehensive view of MoaA kinetics, and determined the rate of the on-pathway 3′,8-cyclization step as 2.7 ± 0.7 s−1. Together with DFT calculations, this observation suggested that the 3′,8-cyclization is accelerated by 6 ∼ 9 orders of magnitude by MoaA. Potential contributions of the active-site amino acid residues, and their potential relationships with human Moco deficiency disease are discussed. This is the first determination of the magnitude of catalytic rate acceleration by a radical SAM enzyme, and provides the foundation for understanding how radical SAM enzymes achieve highly specific radical catalysis.