Characterization of a Radical <i>S</i>‑Adenosyl‑l‑methionine Epimerase, NeoN, in the Last Step of Neomycin B Biosynthesis

Abstract

The last step of neo­mycin biosynthesis is the epimer­ization at C-5‴ of neo­mycin C to give neo­mycin B. A candidate enzyme responsible for the epimer­ization was a putative radical <i>S</i>-adenosyl-l-methionine (SAM) enzyme, NeoN, which is uniquely encoded in the neo­mycin biosynthetic gene cluster and remained an unassigned protein in the neo­mycin biosynthesis. The reconstituted and reduced NeoN showed the expected epimer­ization activity in the presence of SAM. In the epimer­ization, 1 equiv of SAM was consumed to convert neo­mycin C into neo­mycin B. The site of neo­mycin C reactive toward epimer­ization was clearly confirmed to be C-5‴ by detecting the incorporation of a deuterium atom from the deuterium oxide-based buffer solution. Further, alanine scanning of the NeoN cysteine residues revealed that C249 is a critical amino acid residue that provides a hydrogen atom to complete the epimer­ization. Furthermore, electron paramagnetic resonance analysis of the C249A variant in the presence of SAM and neo­mycin C revealed that a radical intermediate is generated at the C-5‴ of neo­mycin C. Therefore, the present study clearly illustrates that the epimer­ization of neo­mycin C to neo­mycin B is catalyzed by a unique radical SAM epimerase NeoN with a radical reaction mechanism