Anderson Karen S
Departments of Pharmacology and Molecular Biophysics and Biochemistry, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06520-8066,USA.
Protein Eng Des Sel. 2017 Mar 1;30(3):253-261. doi: 10.1093/protein/gzx004.
Most species, such as humans, have monofunctional forms of thymidylate synthase (TS) and dihydrofolate reductase (DHFR) that are key folate metabolism enzymes making critical folate components required for DNA synthesis. In contrast, several parasitic protozoa, including Leishmania major (Lm), Plasmodium falciparum (Pf), Toxoplasma gondii (Tg) and Cryptosporidium hominis (Ch), contain a unique bifunctional thymidylate synthase-dihydrofolate reductase (TS-DHFR) having the two sequential catalytic activities contained on a single polypeptide chain. It has been suggested that the bifunctional nature of the two catalytic activities may enable substrate channeling. The 3D structures for each of these enzymes reveals distinct features for each species. While three of the four species (Pf, Tg and Ch) contain a junctional region linking the two domains, this is lacking in Lm. The Lm and Pf contain N-terminal amino acid extensions. A multidisciplinary approach using structural studies and transient kinetic analyses combined with mutational analysis has investigated the roles of these unique structural features for each enzyme. Additionally, the possibility of substrate channeling behavior was explored. These studies have identified unique, functional regions in both the TS and DHFR domains that govern efficient catalysis for each species. Surprisingly, even though there are structural similarities among the species, each is regulated in a distinct manner. This structural and mechanistic information was also used to exploit species-specific inhibitor design.
大多数物种，如人类，具有单功能形式的胸苷酸合成酶（TS）和二氢叶酸还原酶（DHFR），它们是关键的叶酸代谢酶，可产生DNA合成所需的关键叶酸成分。相比之下，几种寄生原生动物，包括硕大利什曼原虫（Lm）、恶性疟原虫（Pf）、刚地弓形虫（Tg）和人隐孢子虫（Ch），含有一种独特的双功能胸苷酸合成酶 - 二氢叶酸还原酶（TS - DHFR），其两种连续的催化活性存在于一条多肽链上。有人提出，这两种催化活性的双功能性质可能有助于底物通道化。这些酶各自的三维结构揭示了每个物种的独特特征。虽然四个物种中的三个（Pf、Tg和Ch）含有连接两个结构域的连接区域，但Lm中没有。Lm和Pf含有N端氨基酸延伸。一种结合结构研究、瞬态动力学分析和突变分析的多学科方法研究了这些独特结构特征对每种酶的作用。此外，还探索了底物通道化行为的可能性。这些研究在TS和DHFR结构域中都确定了独特的功能区域，这些区域控制着每个物种的高效催化。令人惊讶的是，尽管这些物种之间存在结构相似性，但每种物种的调节方式都不同。这些结构和机制信息也被用于开发物种特异性抑制剂设计。
