title: A single amino acid substitution in Staphylococcus aureus dihydrofolate reductase determines trimethoprim resistance.
abstract: A single amino acid substitution, Phe98 to Tyr98, in dihydrofolate reductase (DHFR) is the molecular origin of trimethoprim (TMP) resistance in Staphylococcus aureus. This active site amino acid substitution was found in all S. aureus TMP-resistant clinical isolates tested. In order to explore the structural role of Tyr98 in TMP-resistance the ternary complexes of the chromosomal S. aureus DHFR (SaDHFR) with methotrexate (MTX) and TMP in the presence of nicotinamide adenine dinucleotide phosphate (NADPH) as well as that of mutant Phe98Tyr DHFR SaDHFR(F98Y) ternary folate-NADPH complex have been determined by X-ray crystallography. Critical evidence concerning the resistance mechanism has also been provided by NMR spectral analyses of 15N-labelled TMP in the ternary complexes of both wild-type and mutant enzyme. These studies show that the mutation results in loss of a hydrogen bond between the 4-amino group of TMP and the carbonyl oxygen of Leu5. This mechanism of resistance is predominant in both transferable plasmid-encoded and non-transferable chromosomally encoded resistance. Knowledge of the resistance mechanism at a molecular level could help in the design of antibacterials active against multi-resistant Staphylococcus aureus (MRSA), one of todays most serious problems in clinical infectology.
authors.affiliation: F. Hoffmann-La Roche Ltd, Pharma Preclinical Research Department, Basel, Switzerland.
authors.forename: G E
authors.initials: GE
authors.lastname: Dale
authors.forename: C
authors.initials: C
authors.lastname: Broger
authors.forename: A
authors.initials: A
authors.lastname: D'Arcy
authors.forename: P G
authors.initials: PG
authors.lastname: Hartman
authors.forename: R
authors.initials: R
authors.lastname: DeHoogt
authors.forename: S
authors.initials: S
authors.lastname: Jolidon
authors.forename: I
authors.initials: I
authors.lastname: Kompis
authors.forename: A M
authors.initials: AM
authors.lastname: Labhardt
authors.forename: H
authors.initials: H
authors.lastname: Langen
authors.forename: H
authors.initials: H
authors.lastname: Locher
authors.forename: M G
authors.initials: MG
authors.lastname: Page
authors.forename: D
authors.initials: D
authors.lastname: Stüber
authors.forename: R L
authors.initials: RL
authors.lastname: Then
authors.forename: B
authors.initials: B
authors.lastname: Wipf
authors.forename: C
authors.initials: C
authors.lastname: Oefner
mesh_terms: D001665:Binding Sites; D002876:Chromosomes, Bacterial; D018360:Crystallography, X-Ray; D006801:Humans; D008958:Models, Molecular; D008968:Molecular Conformation; D009249:NADP / Q000737:chemistry / Q000378:metabolism; D010649:Phenylalanine; D017354:Point Mutation; D011487:Protein Conformation; D013211:Staphylococcus aureus / Q000201:enzymology* / Q000235:genetics / Q000302:isolation & purification; D013762:Tetrahydrofolate Dehydrogenase / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D014295:Trimethoprim / Q000737:chemistry / Q000378:metabolism; D014296:Trimethoprim Resistance; D014443:Tyrosine
chemical_list: D014443:Tyrosine; D010649:Phenylalanine; D009249:NADP; D014295:Trimethoprim; D013762:Tetrahydrofolate Dehydrogenase