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HygY Is a Twitch Radical SAM Epimerase with Latent Dehydrogenase Activity Revealed upon Mutation of a Single Cysteine Residue.

ABSTRACT: HygY is a SPASM/twitch radical SAM enzyme hypothesized to catalyze the C2'-epimerization of galacamine during the biosynthesis of hygromycin B. This activity is confirmed via biochemical and structural analysis of the derivatized reaction products using chemically synthesized deuterated substrate, high-resolution mass spectrometry and ¹H NMR. Electron paramagnetic resonance spectroscopy of the reduced enzyme is consistent with ligation of two [Fe₄S₄] clusters characteristic of the twitch radical SAM subgroup. HygY catalyzed epimerization proceeds with incorporation of a single solvent Hydron into the talamine product facilitated by the catalytic cysteine-183 residue. Mutation of this cysteine to alanine converts HygY from a C2'-epimerase to an C2'-dehydrogenase with comparable activity. The SPASM/twitch radical SAM enzymes often serve as anaerobic oxidases making the redox-neutral epimerases in this class rather interesting. The discovery of latent dehydrogenase activity in a twitch epimerase may therefore offer new insights into the mechanistic features that distinguish oxidative versus redox-neutral SPASM/twitch enzymes and lead to the evolution of new enzyme activities.

SUBMITTER: Besandre RA

PROVIDER: S-EPMC8611816 | biostudies-literature |

REPOSITORIES: biostudies-literature

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