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dc.contributor.authorBoehr, D. D.
dc.contributor.authorThompson, Paul R
dc.contributor.authorWright, G. D.
dc.date2022-08-11T08:11:00.000
dc.date.accessioned2022-08-23T17:28:28Z
dc.date.available2022-08-23T17:28:28Z
dc.date.issued2001-06-29
dc.date.submitted2015-06-05
dc.identifier.citationJ Biol Chem. 2001 Jun 29;276(26):23929-36. <a href="http://dx.doi.org/10.1074/jbc.M100540200">Link to article on publisher's site</a>. Epub 2001 Feb 27.
dc.identifier.issn0021-9258 (Linking)
dc.identifier.doi10.1074/jbc.M100540200
dc.identifier.urihttp://hdl.handle.net/20.500.14038/50086
dc.description<p>At the time of publication, Paul Thompson was not yet affiliated with UMass Medical School.</p>
dc.description.abstractThe aminoglycoside antibiotic kinases (APHs) constitute a clinically important group of antibiotic resistance enzymes. APHs share structural and functional homology with Ser/Thr and Tyr kinases, yet only five amino acids are invariant between the two groups of enzymes and these residues are all located within the nucleotide binding regions of the proteins. We have performed site-directed mutagenesis on all five conserved residues in the aminoglycoside kinase APH(3')-IIIa: Lys(44) and Glu(60) involved in ATP capture, a putative active site base required for deprotonating the incoming aminoglycoside hydroxyl group Asp(190), and the Mg(2+) ligands Asn(195) and Glu(208), which coordinate two Mg(2+) ions, Mg1 and Mg2. Previous structural and mutagenesis evidence have demonstrated that Lys(44) interacts directly with the phosphate groups of ATP; mutagenesis of invariant Glu(60), which forms a salt bridge with the epsilon-amino group of Lys(44), demonstrated that this residue does not play a critical role in ATP recognition or catalysis. Results of mutagenesis of Asp(190) were consistent with a role in proper positioning of the aminoglycoside hydroxyl during phosphoryl transfer but not as a general base. The Mg1 and Mg2 ligand Asp(208) was found to be absolutely required for enzyme activity and the Mg2 ligand Asn(195) is important for Mg.ATP recognition. The mutagenesis results together with solvent isotope, solvent viscosity, and divalent cation requirements are consistent with a dissociative mechanism of phosphoryl transfer where initial substrate deprotonation is not essential for phosphate transfer and where Mg2 and Asp(208) likely play a critical role in stabilization of a metaphosphate-like transition state. These results lay the foundation for the synthesis of transition state mimics that could reverse aminoglycoside antibiotic resistance in vivo.
dc.language.isoen_US
dc.relation<a href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&list_uids=11279088&dopt=Abstract">Link to Article in PubMed</a>
dc.relation.urlhttp://dx.doi.org/10.1074/jbc.M100540200
dc.subjectAdenosine Triphosphate
dc.subjectAnimals
dc.subjectAnti-Bacterial Agents
dc.subjectBinding Sites
dc.subjectCatalysis
dc.subjectConserved Sequence
dc.subjectDrug Resistance, Microbial
dc.subjectEndopeptidases
dc.subjectKanamycin
dc.subjectKanamycin Kinase
dc.subjectKinetics
dc.subjectMagnesium
dc.subjectMicrobial Sensitivity Tests
dc.subjectModels, Chemical
dc.subjectMutagenesis, Site-Directed
dc.subjectViscosity
dc.subjectBiochemistry
dc.subjectEnzymes and Coenzymes
dc.subjectMedicinal-Pharmaceutical Chemistry
dc.subjectTherapeutics
dc.titleMolecular mechanism of aminoglycoside antibiotic kinase APH(3')-IIIa: roles of conserved active site residues
dc.typeJournal Article
dc.source.journaltitleThe Journal of biological chemistry
dc.source.volume276
dc.source.issue26
dc.identifier.legacycoverpagehttps://escholarship.umassmed.edu/thompson/90
dc.identifier.contextkey7185902
html.description.abstract<p>The aminoglycoside antibiotic kinases (APHs) constitute a clinically important group of antibiotic resistance enzymes. APHs share structural and functional homology with Ser/Thr and Tyr kinases, yet only five amino acids are invariant between the two groups of enzymes and these residues are all located within the nucleotide binding regions of the proteins. We have performed site-directed mutagenesis on all five conserved residues in the aminoglycoside kinase APH(3')-IIIa: Lys(44) and Glu(60) involved in ATP capture, a putative active site base required for deprotonating the incoming aminoglycoside hydroxyl group Asp(190), and the Mg(2+) ligands Asn(195) and Glu(208), which coordinate two Mg(2+) ions, Mg1 and Mg2. Previous structural and mutagenesis evidence have demonstrated that Lys(44) interacts directly with the phosphate groups of ATP; mutagenesis of invariant Glu(60), which forms a salt bridge with the epsilon-amino group of Lys(44), demonstrated that this residue does not play a critical role in ATP recognition or catalysis. Results of mutagenesis of Asp(190) were consistent with a role in proper positioning of the aminoglycoside hydroxyl during phosphoryl transfer but not as a general base. The Mg1 and Mg2 ligand Asp(208) was found to be absolutely required for enzyme activity and the Mg2 ligand Asn(195) is important for Mg.ATP recognition. The mutagenesis results together with solvent isotope, solvent viscosity, and divalent cation requirements are consistent with a dissociative mechanism of phosphoryl transfer where initial substrate deprotonation is not essential for phosphate transfer and where Mg2 and Asp(208) likely play a critical role in stabilization of a metaphosphate-like transition state. These results lay the foundation for the synthesis of transition state mimics that could reverse aminoglycoside antibiotic resistance in vivo.</p>
dc.identifier.submissionpaththompson/90
dc.contributor.departmentDepartment of Biochemistry and Molecular Pharmacology
dc.source.pages23929-36


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