Identifying Latent Enzyme Activities:  Substrate Ambiguity within Modern Bacterial Sugar Kinases^†

The ability of enzymes to catalyze the transformation of multiple, structurally related substrates could empower the natural evolution of new catalytic functions. The prevalence of such substrate ambiguity in modern catalysts, however, is largely unknown. To search for ambiguous sugar kinases, we generated a bacterium incapable of performing the first step of the glycolytic pathway, the phosphorylation of glucose. This organism cannot survive with glucose as its sole source of carbon. Within its genome, we find three DNA sequences that, when transcribed from a powerful extrachromosomal promoter, can complement the auxotrophy of the organism. These sequences contain the nanK, yajF, and ycfX genes. In vitro, the NanK, YajF, and YcfX proteins function as rudimentary glucokinases with ambiguous substrate specificites, displaying k_cat/K_m values for the phosphorylation of glucose that are 10⁴-fold lower than the k_cat/K_m value of endogenous bacterial glucokinase. Our findings suggest that modern genomes harbor a wealth of latent enzyme activities and that extant metabolic pathways are equivocal, in contrast to their usual depiction.