Blasticidin S Biosynthesis Involves a Unique Interplay between a Radical SAM Dehydratase, a Transaminase and an ATP-Grasp Ligase in Order to Avoid a Futile Cycle.

Blasticidin S is a peptidyl nucleoside that blocks protein synthesis in prokaryotes and eukaryotes. It is widely used in molecular biology as a selection agent. Despite its utility, the biosynthetic origin of its 2',3'-unsaturated-4'-aminoacyl-4'-deoxyhexopyranose core is only partially understood. Previously, the radical -adenosylmethionine enzyme BlsE was shown to catalyze dehydration of cytosylglucuronic acid. Herein, this product is shown to be accepted by the pyridoxal phosphate-dependent enzyme BlsH catalyzing an ,-dehydration coupled to amination affording cytosinine, the sugar core of blasticidin S. The ATP-grasp ligase BlsI is identified as the next enzyme catalyzing amide bond formation between cytosinine and -arginine to yield demethylblasticidin S. BlsI accepts various cationic amino acids to produce blasticidin analogs. In addition, a unique interplay between BlsE, BlsH, and BlsI is revealed. BlsE also catalyzes the oxidation of cytosinine to generate an off-pathway Michael acceptor that can be reductively aminated in the presence of BlsH to regenerate cytosinine. In the presence of BlsI, however, cytosinine undergoes ATP driven amidation thereby breaking the futile cycle. Therefore, this study not only completes the description of blasticidin S biosynthesis but also highlights the promiscuity of BlsE and BlsI, which could be harnessed as biocatalysts for sugar modification and peptide ligation.