Sirawaraporn W, Sirawaraporn R, Chanpongsri A, Jacobs W R, Santi D V
Department of Biochemistry, Faculty of Science, Mahidol University, Bangkok, Thailand.
Exp Parasitol. 1991 Feb;72(2):184-90. doi: 10.1016/0014-4894(91)90136-k.
Dihydrofolate reductase (DHFR) from extracts of Mycobacterium smegmatis strain mc2(6) and trimethoprim-resistant mutant mc2(26) was purified to homogeneity. In crude extracts, the specific activity of the enzyme from the trimethoprim resistant strain was comparable to that from the sensitive strain. The DHFR from both sources was purified using affinity chromatography on MTX-Sepharose followed by Mono Q FPLC. The enzyme has an apparent molecular mass of 23 kDa from gel filtration on Sephadex G-100 and from SDS-PAGE. Amino terminal sequence analysis showed homology with DHFRs from a subset of other gram-positive organisms. The purified enzyme from the trimethoprim-sensitive organism exhibited Km values for H2folate and NADPH of 0.68 +/- 0.2 microM and 21 +/- 4 microM, respectively. The Km values for H2folate and NADPH for the enzyme from the drug-resistant organism were 1.8 +/- 0.4 microM and 5.3 +/- 1.5 microM, respectively. A kcat of 4.5 sec-1 was determined for the DHFR from both sources. The enzyme from both sources was competitively inhibited by pyrimethamine and trimethoprim. The Ki value of trimethoprim, for the enzyme from the drug-resistant organism was about six-fold higher than for the enzyme from drug-sensitive strain. Our data suggest that mutation of DHFR contributes to trimethoprim resistance in the mc2(26) strain of M. smegmatis.
从耻垢分枝杆菌mc2(6)菌株和耐甲氧苄啶突变体mc2(26)提取物中纯化出二氢叶酸还原酶（DHFR），使其达到均一状态。在粗提取物中，耐甲氧苄啶菌株中该酶的比活性与敏感菌株相当。使用MTX-琼脂糖亲和层析，随后进行Mono Q快速蛋白质液相色谱，对来自这两种来源的DHFR进行纯化。通过在Sephadex G-100上的凝胶过滤和SDS-聚丙烯酰胺凝胶电泳，该酶的表观分子量为23 kDa。氨基末端序列分析显示与其他一些革兰氏阳性菌的DHFR具有同源性。来自甲氧苄啶敏感菌的纯化酶对二氢叶酸和烟酰胺腺嘌呤二核苷酸磷酸（NADPH）的米氏常数（Km）分别为0.68±0.2微摩尔和21±4微摩尔。来自耐药菌的该酶对二氢叶酸和NADPH的Km值分别为1.8±0.4微摩尔和5.3±1.5微摩尔。测定了来自这两种来源的DHFR的催化常数（kcat）为4.5秒-1。来自这两种来源的酶均受到乙胺嘧啶和甲氧苄啶的竞争性抑制。甲氧苄啶对耐药菌中该酶的抑制常数（Ki）值比对敏感菌中该酶的Ki值高约6倍。我们的数据表明，DHFR的突变导致了耻垢分枝杆菌mc2(26)菌株对甲氧苄啶的耐药性。
