Ma L, Kovacs J A
Critical Care Medicine Department, Warren Grant Magnuson Clinical Center, National Institutes of Health, Bethesda, Maryland, USA.
Antimicrob Agents Chemother. 2000 Nov;44(11):3092-6. doi: 10.1128/AAC.44.11.3092-3096.2000.
Dihydrofolate reductase (DHFR) is the target of trimethoprim (TMP), which has been widely used in combination with sulfa drugs for treatment and prophylaxis of Pneumocystis carinii pneumonia. While the rat-derived P. carinii DHFR has been well characterized, kinetic studies of human-derived P. carinii DHFR, which differs from rat-derived P. carinii DHFR by 38% in amino acid sequence, have not been reported to date. Here we report on the expression and kinetic characterization of the recombinant human-derived P. carinii DHFR. The 618-bp coding sequence of the human-derived P. carinii DHFR gene was expressed in Escherichia coli. As determined by sodium dodecyl sulfate-polyacrylamide gel eletrophoresis, the purified enzyme had a molecular mass of 25 kDa, consistent with that predicted from the DNA sequence. Kinetic analysis showed that the K(m) values for dihydrofolate and NADPH were 2.7 +/- 0.3 and 14.0 +/- 4.3 microM, respectively, which are similar to those reported for rat-derived P. carinii DHFR. Inhibition studies revealed that both TMP and pyrimethamine were poor inhibitors of human-derived P. carinii DHFR, with K(i) values of 0.28 +/- 0.08 and 0.065 +/- 0.005 microM, respectively, while trimetrexate and methotrexate were potent inhibitors, with K(i) values of 0.23 +/- 0.03 and 0.016 +/- 0.004 nM, respectively. The availability of purified recombinant enzyme in large quantities should facilitate the identification of antifolate inhibitors with greater potency and higher selectivity for human-derived P. carinii DHFR.
二氢叶酸还原酶（DHFR）是甲氧苄啶（TMP）的作用靶点，TMP已广泛与磺胺类药物联合用于治疗和预防卡氏肺孢子虫肺炎。虽然大鼠源卡氏肺孢子虫DHFR已得到充分表征，但人源卡氏肺孢子虫DHFR的动力学研究迄今尚未见报道，其人源卡氏肺孢子虫DHFR与大鼠源卡氏肺孢子虫DHFR的氨基酸序列差异为38%。在此，我们报道重组人源卡氏肺孢子虫DHFR的表达及动力学特征。人源卡氏肺孢子虫DHFR基因的618 bp编码序列在大肠杆菌中表达。经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳测定，纯化后的酶分子量为25 kDa，与DNA序列预测的结果一致。动力学分析表明，二氢叶酸和NADPH的K(m)值分别为2.7±0.3和14.0±4.3 μM，这与大鼠源卡氏肺孢子虫DHFR报道的值相似。抑制研究表明，TMP和乙胺嘧啶对人源卡氏肺孢子虫DHFR的抑制作用较弱，K(i)值分别为0.28±0.08和0.065±0.005 μM，而三甲曲沙和甲氨蝶呤是强效抑制剂，K(i)值分别为0.23±0.03和0.016±0.004 nM。大量纯化重组酶的可得性应有助于鉴定对人源卡氏肺孢子虫DHFR具有更高效力和更高选择性的抗叶酸抑制剂。
