Analogues of 8-hydroxy-5-deazaflavin cofactor: relative activity as substrates for 8-hydroxy-5-deazaflavin-dependent NADP+ reductase from Methanococcus vannielii.

Abstract

The 8-hydroxy-5-deazaflavin-dependent NADP+ reductase from Methanococcus vannielii was examined for its ability to catalyze the reduction of a number of 5-deazaflavin analogues of the natural cofactor. Comparison of the kinetic constants revealed certain substrate structure-reactivity relationships for the enzyme. The basic heterocyclic system of the natural cofactor 2,4-dioxopyrimido [4,5-b]quinoline was shown to be the minimum structural requirement since neither riboflavin nor 1,5-dideazariboflavin was reduced by the enzyme. The N-10 side chain of the natural cofactor was shown not to be essential since the enzyme could reduce 8-hydroxy-2,4-dioxopyrimido[4,5-b]quinoline. The study also indicated that there are some steric constraints at C-8 and C-7 with respect to interaction of the cofactor with the enzyme. Specifically, (a) the 8-methoxy derivative, in contrast to the 8-hydroxy compound, was not reduced and (b) the introduction of a substituent at C-7 resulted in a marked decrease in the rate of reduction. The importance of C-5 as the site for the electron entry was suggested by the finding that 5-methyl-deazariboflavin was not reduced. The latter inhibited the reduction of 5-deazariboflavin.