title: Role of aspartate 143 in Escherichia coli tRNA-guanine transglycosylase: alteration of heterocyclic substrate specificity.
abstract: tRNA-guanine transglycosylase (TGT) is a key enzyme involved in the post-transcriptional modification of certain tRNAs in their anticodon wobble positions with queuine. To maintain the correct Watson-Crick base pairing properties of the wobble base (and hence proper translation of the genetic code), TGT must recognize its heterocyclic substrate with high specificity. The X-ray crystal structure of a eubacterial TGT bound to preQ1 [Romier, C., et al. (1996) EMBO J. 15, 2850-2857] suggested that aspartate 143 (Escherichia coli TGT numbering) was involved in heterocyclic substrate recognition. Subsequent mutagenic and computational modeling studies from our lab [Todorov, K. A., et al. (2005) Biophys. J. 89 (3), 1965-1977] provided experimental evidence supporting this hypothesis. Herein, we report further studies probing the differential heterocyclic substrate recognition properties of the aspartate 143 mutant TGTs. Our results are consistent with one of the mutants exhibiting an inversion of substrate recognition preference (xanthine vs guanine) relative to that of the wild type, as evidenced by Km values. This confirms the key role of aspartate 143 in maintaining the anticodon identities of the queuine-containing tRNAs and suggests that TGT mutants could be developed that would alter the tRNA wobble base base pairing properties.
authors.affiliation: Department of Medicinal Chemistry, College of Pharmacy, University of Michigan, Ann Arbor, Michigan 48109-1065, USA.
authors.forename: Katherine Abold
authors.initials: KA
authors.lastname: Todorov
authors.forename: George A
authors.initials: GA
authors.lastname: Garcia
mesh_terms: D019943:Amino Acid Substitution / Q000235:genetics; D000926:Anticodon / Q000737:chemistry; D001224:Aspartic Acid / Q000737:chemistry* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics; D019271:Hypoxanthine / Q000737:chemistry; D007700:Kinetics; D009154:Mutation; D010430:Pentosyltransferases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics; D012694:Serine / Q000235:genetics; D013379:Substrate Specificity / Q000235:genetics; D013912:Threonine / Q000235:genetics; D019820:Xanthine / Q000737:chemistry
chemical_list: D000926:Anticodon; D029968:Escherichia coli Proteins; D019820:Xanthine; D019271:Hypoxanthine; D013912:Threonine; D001224:Aspartic Acid; D012694:Serine; D010430:Pentosyltransferases; C020837:queuine tRNA-ribosyltransferase