Divergent evolution of function in the ROK sugar kinase superfamily: role of enzyme loops in substrate specificity

The d-allose and N-acetyl-d-mannosamine kinases of Escherichia coli K-12 are divergent members of the functionally diverse ROK (repressor, open reading frame, kinase) superfamily. Previous work in our laboratory has demonstrated that AlsK and NanK possess weak phosphoryl transfer activity toward the alternate substrate d-glucose. To gain insight into the evolutionary mechanisms that fuel the specialization of individual enzyme function, experimental laboratory evolution was conducted to improve the glucokinase activities of AlsK and NanK. Error-prone PCR was combined with in vivo functional selection in a glucokinase-deficient bacterium to identify four independent single nucleotide substitutions in the alsK and nanK genes that improve the glucokinase activity of each enzyme. The most advantageous substitutions, L84P in NanK and A73G in AlsK, enhance the kcat/Km values for phosphoryl transfer to glucose by 12-fold and 60-fold, respectively. Both substitutions co-localize to a variable loop region located between the fourth beta-sheet and the second alpha-helix of the ROK scaffold. A multiple sequence alignment of diverse ROK family members reveals that the A73G substitution in AlsK recapitulates a conserved glycine residue present in many ROK proteins, including some transcriptional repressors. Steady-state kinetic analyses of the selected AlsK and NanK variants demonstrate that their native activities toward d-allose and N-acetyl-d-mannosamine are largely unaffected by the glucokinase-enhancing substitutions. Substrate specificity profiling reveals that the A73G AlsK and L84P NanK variants display systematic improvements in the kcat/Km values for a variety of nonnative carbohydrates. This finding is consistent with an evolutionary process that includes the formation of intermediates possessing relaxed substrate specificities during the initial steps of enzyme functional divergence.