Department of Biochemistry and Center of Excellence in Protein Structure & Function, Faculty of Science, Mahidol University, Rama 6 Road Bangkok 10400, Thailand.
Malar J. 2012 Jun 12;11:194. doi: 10.1186/1475-2875-11-194.
There is an urgent need for the discovery of new anti-malarial drugs. Thus, it is essential to explore different potential new targets that are unique to the parasite or that are required for its viability in order to develop new interventions for treating the disease. Plasmodium serine hydroxymethyltransferase (SHMT), an enzyme in the dTMP synthesis cycle, is a potential target for such new drugs, but convenient methods for producing and assaying the enzyme are still lacking, hampering the ability to screen inhibitors.
Production of recombinant Plasmodium falciparum SHMT (PfSHMT) and Plasmodium vivax SHMT (PvSHMT), using auto-induction media, were compared to those using the conventional Luria Bertani medium with isopropyl thio-β-D-galactoside (LB-IPTG) induction media. Plasmodium SHMT activity, kinetic parameters, and response to inhibitors were measured spectrophotometrically by coupling the reaction to that of 5,10-methylenetetrahydrofolate dehydrogenase (MTHFD). The identity of the intermediate formed upon inactivation of Plasmodium SHMTs by thiosemicarbazide was investigated by spectrophotometry, high performance liquid chromatography (HPLC), and liquid chromatography-mass spectrometry (LC-MS). The active site environment of Plasmodium SHMT was probed based on changes in the fluorescence emission spectrum upon addition of amino acids and folate.
Auto-induction media resulted in a two to three-fold higher yield of Pf- and PvSHMT (7.38 and 29.29 mg/L) compared to that produced in cells induced in LB-IPTG media. A convenient spectrophotometric activity assay coupling Plasmodium SHMT and MTHFD gave similar kinetic parameters to those previously obtained from the anaerobic assay coupling SHMT and 5,10-methylenetetrahydrofolate reductase (MTHFR); thus demonstrating the validity of the new assay procedure. The improved method was adopted to screen for Plasmodium SHMT inhibitors, of which some were originally designed as inhibitors of malarial dihydrofolate reductase. Plasmodium SHMT was slowly inactivated by thiosemicarbazide and formed a covalent intermediate, PLP-thiosemicarbazone.
Auto-induction media offers a cost-effective method for the production of Plasmodium SHMTs and should be applicable for other Plasmodium enzymes. The SHMT-MTHFD coupled assay is equivalent to the SHMT-MTHFR coupled assay, but is more convenient for inhibitor screening and other studies of the enzyme. In addition to inhibitors of malarial SHMT, the development of species-specific, anti-SHMT inhibitors is plausible due to the presence of differential active sites on the Plasmodium enzymes.
迫切需要发现新的抗疟药物。因此，探索寄生虫特有的或寄生虫生存所需的不同潜在新靶标对于开发治疗疾病的新干预措施至关重要。磷酸丝氨酸羟甲基转移酶（SHMT）是 dTMP 合成循环中的一种酶，是此类新药的潜在靶标，但仍缺乏方便的酶生产和检测方法，这阻碍了抑制剂的筛选能力。
比较了使用自动诱导培养基和常规的含异丙基硫代-β-D-半乳糖苷（LB-IPTG）的 Luria Bertani 培养基（LB-IPTG）诱导培养来生产重组恶性疟原虫 SHMT（PfSHMT）和间日疟原虫 SHMT（PvSHMT）。通过将反应与 5,10-亚甲基四氢叶酸脱氢酶（MTHFD）偶联来分光光度法测定疟原虫 SHMT 的活性、动力学参数和对抑制剂的反应。通过分光光度法、高效液相色谱法（HPLC）和液相色谱-质谱法（LC-MS）研究了半硫脲嘧啶使疟原虫 SHMT 失活后形成的中间产物。基于添加氨基酸和叶酸后荧光发射光谱的变化，探测了疟原虫 SHMT 的活性部位环境。
与在 LB-IPTG 培养基中诱导产生的细胞相比，自动诱导培养基使 Pf-和 PvSHMT（7.38 和 29.29 mg/L）的产量提高了两到三倍。一种方便的分光光度活性测定法将 Plasmodium SHMT 和 MTHFD 偶联，得到的动力学参数与以前从厌氧测定法将 SHMT 和 5,10-亚甲基四氢叶酸还原酶（MTHFR）偶联获得的参数相似；从而证明了新测定程序的有效性。采用改进的方法筛选疟原虫 SHMT 抑制剂，其中一些最初是作为疟原虫二氢叶酸还原酶抑制剂设计的。半硫脲嘧啶使疟原虫 SHMT 缓慢失活并形成共价中间产物，PLP-半硫脲嘧啶。
自动诱导培养基为 Plasmodium SHMT 的生产提供了一种具有成本效益的方法，并且应该适用于其他疟原虫酶。SHMT-MTHFD 偶联测定与 SHMT-MTHFR 偶联测定等效，但更便于抑制剂筛选和酶的其他研究。除了抗疟 SHMT 抑制剂外，由于疟原虫酶的活性部位存在差异，开发针对特定物种的抗 SHMT 抑制剂也是合理的。
