Alfadhli S, Rathod P K
Department of Biology, The Catholic University of America, Washington DC 20064, USA.
Mol Biochem Parasitol. 2000 Oct;110(2):283-91. doi: 10.1016/s0166-6851(00)00282-6.
The global emergence of drug-resistant malarial parasites necessitates identification and characterization of novel drug targets. Three reactions are involved in methylenetetrahydrofolate recycling: Thymidylate synthase (TS), dihydrofolate reductase (DHFR), and serine hydroxymethyltransferase (SHMT). Malarial bifunctional DHFR-TS is a well-studied, important target of established drugs such as pyrimethamine and cycloguanil. In sharp contrast, malarial SHMT remains largely uncharacterized. In the present study, a Plasmodium falciparum SHMT coding region was characterized. It had 1603 bp including two introns near the 5'-end of the gene: one 118 bp intron immediately after the start methionine and a 159 bp intron after an additional 34 amino acids. The three exons together coded for a 442 amino acid protein with 38-47% identity to SHMT sequences from other species. Expression of malarial SHMT coding sequence (minus the introns) into glyA mutants of Escherichia coli relieved glycine auxotrophy and permitted direct assay of SHMT catalytic activity in bacterial cell lysates. This is the first SHMT cloned and expressed from a protozoan parasite. The molecular tools developed in this study will be useful for developing potential antimalarials directed at SHMT.
全球耐药疟原虫的出现使得鉴定和表征新型药物靶点成为必要。亚甲基四氢叶酸循环涉及三种反应：胸苷酸合成酶（TS）、二氢叶酸还原酶（DHFR）和丝氨酸羟甲基转移酶（SHMT）。疟原虫双功能DHFR-TS是已得到充分研究的重要靶点，如乙胺嘧啶和环氯胍等现有药物都是针对该靶点。与之形成鲜明对比的是，疟原虫SHMT在很大程度上仍未得到充分表征。在本研究中，对恶性疟原虫SHMT编码区进行了表征。它有1603 bp，包括基因5'端附近的两个内含子：一个118 bp的内含子紧跟在起始甲硫氨酸之后，另一个159 bp的内含子在额外34个氨基酸之后。这三个外显子共同编码一个442个氨基酸的蛋白质，与其他物种的SHMT序列有38 - 47%的同源性。将疟原虫SHMT编码序列（不含内含子）导入大肠杆菌的glyA突变体中，可缓解甘氨酸营养缺陷，并能直接检测细菌细胞裂解物中的SHMT催化活性。这是首次从原生动物寄生虫中克隆并表达的SHMT。本研究中开发的分子工具将有助于开发针对SHMT的潜在抗疟药物。
