The biosynthesis of blasticidin S has drawn attention due to the participation of the radical S-adenosyl methionine (SAM) enzyme BlsE. The original assignment of BlsE as a radical-mediated, redox-neutral decarboxylase is unusual because this reaction appears to serve no biosynthetic purpose and would need to be reversed by a subsequent carboxylation step. Furthermore, with the exception of BlsE, all other radical SAM decarboxylases reported to date are oxidative in nature. Careful analysis of the BlsE reaction, however, demonstrates that BlsE is not a decarboxylase but instead a lyase that catalyzes the dehydration of cytosylglucuronic acid (CGA) to form cytosyl-4′-keto-3′-deoxy-d-glucuronic acid, which can rapidly decarboxylate nonenzymatically in vitro. Analysis of substrate isotopologs, fluorinated analogues, as well as computational models based on X-ray crystal structures of the BlsE·SAM (2.09 Å) and BlsE·SAM·CGA (2.62 Å) complexes suggests that BlsE catalysis likely proceeds via direct elimination of water from the CGA C4′ α-hydroxyalkyl radical as opposed to 1,2-migration of the C3′-hydroxyl prior to dehydration. Biosynthetic and mechanistic implications of the revised assignment of BlsE are discussed.

Spectinomycin is a dioxane-bridged, tricyclic aminoglycoside produced by Streptomyces spectabilis ATCC 27741. While the spe biosynthetic gene cluster for spectinomycin has been reported, the chemistry underlying construction of the dioxane ring is unknown. The twitch radical SAM enzyme SpeY from the spe cluster is shown here to catalyze dehydrogenation of the C2′ alcohol of (2′R,3′S)-tetrahydrospectinomycin to yield (3′S)dihydrospectinomycin as a likely biosynthetic intermediate. This reaction is radical-mediated and initiated via H atom abstraction from C2′ of the substrate by the 5′-deoxyadenosyl radical equivalent generated upon reductive cleavage of SAM. Crystallographic analysis of the ternary Michaelis complex places serine-183 adjacent to C2′ of the bound substrate opposite C5′ of SAM. Mutation of this residue to cysteine converts SpeY to the corresponding C2′ epimerase mirroring the opposite phenomenon observed in the homologous twitch radical SAM epimerase HygY from the hygromycin B biosynthetic pathway. Phylogenetic analysis suggests a relatively recent evolutionary branching of putative twitch radical SAM epimerases bearing homologous cysteine residues to generate the SpeY clade of enzymes.