RESUMO
The last step of neomycin biosynthesis is the epimerization at C-5â´ of neomycin C to give neomycin B. A candidate enzyme responsible for the epimerization was a putative radical S-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-5â´ 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-5â´ 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.
