posted on 2007-11-27, 00:00authored byMioara Larion, Lauren B. Moore, Steven M. Thompson, Brian G. Miller
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 β-sheet and the second α-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.