CofE catalyzes the addition of two Glutamates to F420-0 in F420 coenzyme biosynthesis in Methanococcus jannaschii

The protein product of the Methanococcus jannaschii MJ0768 gene has been expressed in Escherichia coli, purified to homogeneity, and shown to catalyze the GTP-dependent addition of two L-glutamates to the L-lactyl phosphodiester of 7,8-didemethyl-8-hydroxy-5-deazariboflavin (F-420-0) to form F-420-0-glutamyl-glutamate (F-420-2). Since the reaction is the fifth step in the biosynthesis of coenzyme F-420, the enzyme has been designated as CofE, the product of the cofE gene. Gel filtration chromatography indicates CofE is a dimer. The enzyme has no recognized sequence similarity to any previously characterized proteins. The enzyme has an absolute requirement for a divalent metal ion and a monovalent cation. Among the metal ions tested, a mixture of Mn2+, Mg2+, and K+ is the most effective. CofE catalyzes amide bond formation with the cleavage of GTP to GDP and inorganic phosphate, likely involving the activation of the free carboxylate group of F-420-0 to give an acyl phosphate intermediate. Evidence for the occurrence of this intermediate is presented. A reaction mechanism for the enzyme is proposed and compared with other members of the ADP-forming amide bond ligase family.