Dale G E, Broger C, D'Arcy A, Hartman P G, DeHoogt R, Jolidon S, Kompis I, Labhardt A M, Langen H, Locher H, Page M G, Stüber D, Then R L, Wipf B, Oefner C
F. Hoffmann-La Roche Ltd, Pharma Preclinical Research Department, Basel, Switzerland.
J Mol Biol. 1997 Feb 14;266(1):23-30. doi: 10.1006/jmbi.1996.0770.
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.
二氢叶酸还原酶（DHFR）中单个氨基酸的替换，即苯丙氨酸98突变为酪氨酸98，是金黄色葡萄球菌对甲氧苄啶（TMP）耐药的分子起源。在所有测试的金黄色葡萄球菌TMP耐药临床分离株中均发现了这一活性位点氨基酸替换。为了探究酪氨酸98在TMP耐药中的结构作用，通过X射线晶体学确定了染色体金黄色葡萄球菌DHFR（SaDHFR）与甲氨蝶呤（MTX）和TMP在烟酰胺腺嘌呤二核苷酸磷酸（NADPH）存在下的三元复合物，以及突变型苯丙氨酸98酪氨酸DHFR SaDHFR（F98Y）的叶酸 - NADPH三元复合物。对野生型和突变型酶的三元复合物中15N标记的TMP进行核磁共振光谱分析，也提供了有关耐药机制的关键证据。这些研究表明，该突变导致TMP的4 - 氨基与亮氨酸5的羰基氧之间的氢键丧失。这种耐药机制在可转移质粒编码和非转移染色体编码的耐药中均占主导地位。在分子水平上了解耐药机制有助于设计对多重耐药金黄色葡萄球菌（MRSA）有效的抗菌药物，MRSA是当今临床感染学中最严重的问题之一。
