A SINGLE AMINO-ACID SUBSTITUTION IN STAPHYLOCOCCUS-AUREUS DIHYDROFOLATE-REDUCTASE DETERMINES TRIMETHOPRIM RESISTANCE

A single amino acid substitution, Phe98 to Tyr98, in dihydrofolate red uctase (DHFR) is the molecular origin of trimethoprim (TMP) resistance in Staphylococcus aureus. This active site amino acid substitution wa s found in all S. aureus TMP-resistant clinical isolates tested. In or der to explore the structural role of Tyr98 in TMP-resistance the tern ary complexes of the chromosomal S. aureus DHFR (SaDHFR) with methotre xate (MTX) and TMP in the presence of nicotinamide adenine dinucleotid e phosphate (NADPH) as well as that of mutant Phe98Tyr DHFR SaDHFR(F98 Y) ternary folate-NADPH complex have been determined by X-ray crystall ography. Critical evidence concerning the resistance mechanism has als o been provided by NMR spectral analyses of N-15-labelled TMP in the t ernary complexes of both wild-type and mutant enzyme. These studies sh ow 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 resis tance mechanism at a molecular level could help in the design of antib acterials active against multi-resistant Staphylococcus aureus (MRSA), one of todays most serious problems in clinical infectology. (C) 1997 Academic Press Limited.