title: Bulky "gatekeeper" residue changes the cosubstrate specificity of aminoglycoside 2''-phosphotransferase IIa.
abstract: The aminoglycoside 2"-phosphotransferases APH(2")-IIa and APH(2")-IVa can utilize ATP and GTP as cosubstrates, since both enzymes possess overlapping but discrete structural templates for ATP and GTP binding. APH(2″)-IIIa uses GTP exclusively, because its ATP-binding template is blocked by a bulky tyrosine "gatekeeper" residue. Replacement of the "gatekeeper" residues M85 and F95 in APH(2")-IIa and APH(2")-IVa, respectively, by tyrosine does not significantly change the antibiotic susceptibility profiles produced by the enzymes. In APH(2")-IIa, M85Y substitution results in an ~10-fold decrease in the K(m) value of GTP and an ~320-fold increase in the K(m) value of ATP. In APH(2")-IVa, F95Y substitution results in a modest decrease in the K(m) values of both GTP and ATP. Structural analysis indicates that in the APH(2")-IIa M85Y mutant, tyrosine blocks access of ATP to the correct position in the binding site, while the larger nucleoside triphosphate (NTP)-binding pocket of the APH(2")-IVa F95Y mutant allows the tyrosine to move away, thus giving access to the ATP-binding template.
authors.affiliation: Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana, USA.
authors.forename: Monolekha
authors.initials: M
authors.lastname: Bhattacharya
authors.forename: Marta
authors.initials: M
authors.lastname: Toth
authors.forename: Clyde A
authors.initials: CA
authors.lastname: Smith
authors.forename: Sergei B
authors.initials: SB
authors.lastname: Vakulenko
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism*; D019943:Amino Acid Substitution; D000617:Aminoglycosides / Q000494:pharmacology*; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics; D001665:Binding Sites; D004926:Escherichia coli / Q000737:chemistry / Q000187:drug effects* / Q000201:enzymology / Q000235:genetics; D005798:Genes, Bacterial; D005822:Genetic Vectors; D006160:Guanosine Triphosphate / Q000737:chemistry; D008826:Microbial Sensitivity Tests; D016297:Mutagenesis, Site-Directed; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000737:chemistry* / Q000235:genetics; D017433:Protein Structure, Secondary; D013379:Substrate Specificity; D014443:Tyrosine / Q000737:chemistry
chemical_list: D000617:Aminoglycosides; D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D014443:Tyrosine; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D017853:Phosphotransferases (Alcohol Group Acceptor); C048564:aminoglycoside 2''-phosphotransferase