Structure and kinetics assays of recombinant Schistosoma mansoni dihydrofolate reductase
Structure and kinetics assays of recombinant Schistosoma mansoni dihydrofolate reductase
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DOI：
10.1016/j.actatropica.2017.03.007
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发表时间：
2017-06-01
期刊：
影响因子：
2.7
通讯作者：
Pereira, Humberto D'Muniz
中科院分区：
文献类型：
--
作者：
Balasco Serrao, Vitor Hugo;Romanello, Larissa;Pereira, Humberto D'Muniz
曼氏血吸虫具有嘧啶生物合成的所有途径，其中胸苷酸循环参与者二氢叶酸还原酶（DHFR）是核苷酸代谢获得能量和结构核酸所必需的。因此，DHFRs已被广泛建议作为治疗传染病的治疗靶点。在本研究中，我们以异源方式表达重组SmDHFR，以获得其结构、生化和动力学信息。重组SmDHFR在1.95埃分辨率下的x射线衍射显示其结构具有典型的DHFR折叠。采用等温滴定量热法测定了NADP（+）和二氢叶酸的动力学常数。此外，使用商业叶酸类似物甲氨蝶呤和氨蝶呤进行抑制试验；这些类似物被认为是叶酸的竞争对手，并被用作癌症和自身免疫性疾病的化疗药物。本研究提供的信息可能对未来新药的发现和理解曼氏梭菌代谢途径的这些代谢步骤有用，从而有助于我们理解寄生虫代谢的这些基本途径的功能。(C) 2017 Elsevier B.V.版权所有
The parasite Schistosoma mansoni possesses all pathways for pyrimidine biosynthesis, in which dihydrofolate reductase (DHFR), thymidylate cycle participants, is essential for nucleotide metabolism to obtain energy and structural nucleic acids. Thus, DHFRs have been widely suggested as therapeutic targets for the treatment of infectious diseases. In this study, we expressed recombinant SmDHFR in a heterologous manner to obtain structural, biochemical and kinetic information. X-ray diffraction of recombinant SmDHFR at 1.95 angstrom resolution showed that the structure exhibited the canonical DHFR fold. Isothermal titration calorimetry was used to determine the kinetic constants for NADP(+) and dihydrofolate. Moreover, inhibition assays were performed using the commercial folate analogs methotrexate and aminopterin; these analogs are recognized as folate competitors and are used as chemotherapeutic agents in cancer and autoimmune diseases. This study provides information that may prove useful for the future discovery of novel drugs and for understanding these metabolic steps from this pathway of S. mansoni, thus aiding in our understanding of the function of these essential pathways for parasite metabolism. (C) 2017 Elsevier B.V. All rights reserved.