Gamarro F, Yu P L, Zhao J, Edman U, Greene P J, Santi D
Department of Biochemistry and Biophysics, University of California, San Francisco 94143, USA.
Mol Biochem Parasitol. 1995 Jun;72(1-2):11-22. doi: 10.1016/0166-6851(95)00059-a.
The gene encoding the bifunctional dihydrofolate reductase (DHFR) and thymidylate synthase (TS) of Trypanosoma brucei brucei has been isolated and expressed in Escherichia coli, and the enzyme has been purified and characterized. The coding sequence of the DHFR-TS is 1581 nt, encoding a 527-amino-acid protein of 58,505 Da. The gene was expressed under control of the trc promoter in pKK233-2. The resulting expression plasmid conferred trimethoprim resistance to E. coli DH5 alpha and complemented the TS deficiency in chi 2913recA cells indicating the presence of active DHFR and TS. DHFR-TS was purified by methotrexate-Sepharose chromatography. In addition to the full-length enzyme, the purified enzyme contained 31 and 31.5-kDa forms of the enzyme that cross-reacted with anti-L. major DHFR-TS antibodies; one was truncated at the N- and C termini, and the other at only the C terminus. Despite the presence of sufficient TS for complementation, TS activity was not detectable in the crude extract or in the final purified enzyme preparation. Although the majority of the enzyme appears to be full length, it is possible that the TS domain has been degraded by one of more residues, which would inactivate the ability to synthesize thymidylate. Kinetic analysis of DHFR yielded kcat and Km values similar to those of related enzymes. The T. brucei DHFR has Ki values for antimicrobial antifolates pyrimethamine and trimethoprim which are significantly lower than the closely related T. cruzi or L. major DHFRs or than human DHFR.
布氏布氏锥虫双功能二氢叶酸还原酶（DHFR）和胸苷酸合成酶（TS）的编码基因已被分离并在大肠杆菌中表达，该酶已被纯化并进行了特性分析。DHFR-TS的编码序列为1581个核苷酸，编码一个527个氨基酸、分子量为58,505道尔顿的蛋白质。该基因在pKK233-2的trc启动子控制下表达。所得表达质粒赋予大肠杆菌DH5α对甲氧苄啶的抗性，并补充了chi 2913recA细胞中的TS缺陷，表明存在活性DHFR和TS。通过甲氨蝶呤-琼脂糖凝胶层析法纯化DHFR-TS。除了全长酶外，纯化后的酶还包含与抗大利什曼原虫DHFR-TS抗体发生交叉反应的31 kDa和31.5 kDa形式的酶；一种在N端和C端均被截断，另一种仅在C端被截断。尽管存在足以用于互补的TS，但在粗提物或最终纯化的酶制剂中均未检测到TS活性。虽然大多数酶似乎是全长的，但TS结构域有可能已被一个或多个残基降解，这将使其合成胸苷酸的能力失活。对DHFR的动力学分析得出的kcat和Km值与相关酶相似。布氏锥虫DHFR对抗微生物抗叶酸剂乙胺嘧啶和甲氧苄啶的Ki值明显低于密切相关的克氏锥虫或大利什曼原虫DHFRs，也低于人类DHFR。
