title: Identifying latent enzyme activities: substrate ambiguity within modern bacterial sugar kinases.
abstract: 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 10(4)-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.
authors.affiliation: Department of Biochemistry, University of Wisconsin-Madison, Madison, Wisconsin 53706-1544, USA.
authors.forename: Brian G
authors.initials: BG
authors.lastname: Miller
authors.forename: Ronald T
authors.initials: RT
authors.lastname: Raines
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D015723:Gene Library; D005941:Glucokinase / Q000235:genetics / Q000378:metabolism*; D005947:Glucose / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D010766:Phosphorylation; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D016601:RNA-Binding Proteins; C473713:YajQ protein, E coli; D017853:Phosphotransferases (Alcohol Group Acceptor); D005941:Glucokinase; C018765:N-acylmannosamine kinase; D005947:Glucose