What question did this study set out to answer?

This research investigates how a single mutation in the BlsE enzyme can alter its catalytic activity and product specificity.

February 13, 2026

The Radical SAM Enzyme BlsE Can Operate as a Dehydratase, Dehydrogenase, or Epimerase upon Mutation of a Single Active Site Serine Residue

Key Points

This research investigates how a single mutation in the BlsE enzyme can alter its catalytic activity and product specificity.
Mutagenesis of the BlsE enzyme focusing on Ser275 residue
Stoichiometric analysis of enzymatic reactions
Comparison of wild-type and mutant BlsE functions
Assessment of reaction products produced by different BlsE mutants
The BlsE-S275A mutant shows a bias toward dehydrogenation and exhibits reversed stereoselectivity in dehydration products.
The S275C mutant functions as an epimerase and accepts various cytosylglucoronic acid analogues.
Different products (dehydration, dehydrogenation, epimerization) arise from the same substrate radical intermediate.

Abstract

Radical S-adenosylmethionine (SAM) enzymes are versatile biocatalysts catalyzing a broad range of radical-mediated transformations. While the native enzymes typically catalyze reactions that yield specific products of biosynthetic relevance, structural variation of the active site or the substrate itself can perturb this tightly controlled process, leading to changes in the reaction outcome. BlsE is a twitch radical SAM diol-dehydratase involved in the biosynthesis of the fungicide blasticidin S. Mutagenesis and stoichiometric analysis of the BlsE-catalyzed reaction identify Ser275 as critical to the catalytic outcome despite a lack of direct interactions with the bound substrate. The BlsE-S275A mutant is biased toward dehydrogenation instead of dehydration, and the residual dehydration product produced by this mutant exhibits a reversed protonation stereoselectivity compared to the wild-type enzyme. Moreover, the residue at position 275 is poised for H atom transfer if swapped for a cysteine such that the S275C mutant operates as a C4' epimerase that accepts at least eight different cytosylglucoronic acid analogues. These results indicate that a common substrate radical intermediate can be quenched in different ways to afford dehydration, dehydrogenation, and epimerization products, thereby demonstrating the ease with which radical SAM enzymes can gain new functions. Importantly, a comparison with other radical SAM diol-dehydrogenases and diol-dehydratases also reveals the importance of active site preorganization in radical-mediated reactions to facilitate specific reaction outcomes over others. Such overarching mechanistic principles bring us one step closer to engineering biocatalysts based on radical SAM chemistry.

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