The enzyme, isolated from the bacterium Micromonospora echinospora, has a single [4Fe-4S] cluster per monomer. It is a radical S-adenosyl-L-methionine (SAM) enzyme with a methylcob(III)alamin cofactor. The enzyme uses two molecues of SAM for the reaction. One molecule forms a 5'-deoxyadenosyl radical, while the other is used to methylate the cobalamin cofactor. It catalyses methylation of the 6'-carbon of gentamicin X2 (GenX2) to produce genetricin (G418) during the biosynthesis of gentamicins. The 6'-pro-R-hydrogen atom of GenX2 is stereoselectively abstracted by the 5'-deoxyadenosyl radical and methylation occurs with retention of configuration at C6'. The regeneration of cob(I)alamin from cob(III)alamin is carried out with an as yet unidentified electron donor.
Reaction Catalyzed by GenK, a Cobalamin-Dependent Radical S-Adenosyl-l-methionine Methyltransferase in the Biosynthetic Pathway of Gentamicin, Proceeds with Retention of Configuration.