Probing the role of parasite-specific, distant structural regions on communication and catalysis in the bifunctional thymidylate synthase-dihydrofolate reductase from Plasmodium falciparum
Probing the role of parasite-specific, distant structural regions on communication and catalysis in the bifunctional thymidylate synthase-dihydrofolate reductase from Plasmodium falciparum
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DOI：
10.1021/bi701624u
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发表时间：
2008-02-05
期刊：
影响因子：
2.9
通讯作者：
Anderson, Karen S.
中科院分区：
文献类型：
--
作者：
Dasgupta, Tina;Anderson, Karen S.
恶性疟原虫胸苷酸合成酶-二氢叶酸还原酶（TS-DHFR）是核苷酸生物合成中的必需酶，也是疟疾有效的分子药物靶点。由于恶性疟原虫TS和DHFR与它们的人类对应物高度同源，现有的活性位点抗叶酸药物可能具有剂量限制性毒性。在人类中，TS和DHFR是两种不同的蛋白质。然而，在恶性疟原虫中，TS-DHFR是双功能的，TS和DHFR活性位点都在酶的单个多肽链上。因此，恶性疟原虫TS-DHFR含有可能调节催化作用的独特远端或非活性区域：（1）N末端尾和（2）将DHFR拴系到TS的接头区域，并编码与DHFR活性位点形成关键静电相互作用的交叉螺旋。这些非活性位点在双功能恶性疟原虫TS-DHFR中的作用尚不清楚。我们报告了全长野生型恶性疟原虫TS-DHFR酶的第一个深入的、预稳态动力学表征，并通过突变分析探讨了远端非活性区域的作用。我们表明WT恶性疟原虫TS-DHFR酶的总体限速步骤是TS催化。我们进一步表明，如果TS处于活化（配体）构象，则DHFR速率为2倍活化，在WT酶中为60 s（-1）至130 s（-1）。如果DHFR处于活化的配体结合构象，则TS速率也被近似1.5倍地活化。接头区的突变既不影响催化速率，也不影响结构域-结构域通信。N-末端尾部的缺失，虽然在远离活性位点的位置，但使DHFR单一速率和双功能TS-DHFR速率降低2倍。TS配体对DHFR速率的2倍活化保持完整，尽管即使活化的N-末端突变体也仅具有WT酶的DHFR活性的一半。然而，TS活性位点和DHFR配体之间的相互通信在N-末端突变体中受损。令人惊讶的是，在大型利什曼原虫TS-DHFR中缺失类似的N-末端尾部导致DHFR速率从近似14 s（-1）增加到近似40 s（-1）。总之，我们的研究结果表明，在恶性疟原虫TS-DHFR中，结构域-结构域通信和催化的非活性位点调节之间存在复杂的相互作用。此外，每个寄生TS-DHFR由独特的机制激活，由其非活性部位区域调节。最后，我们的研究表明，恶性疟原虫TS-DHFR的N-末端尾部是一个高度选择性的，新的目标，为潜在的抗叶酸剂的发展在疟疾。
Plasmodium falciparum thymidylate synthase- dihydrofolate reductase (TS-DHFR) is an essential enzyme in nucleotide biosynthesis and a validated molecular drug target in malaria. Because P. falciparum TS and DHFR are highly homologous to their human counterparts, existing active-site antifolate drugs can have dose-limiting toxicities. In humans, TS and DHFR are two separate proteins. In P. falciparum, however, TS-DHFR is bifunctional, with both TS and DHFR active sites on a single polypeptide chain of the enzyme. Consequently, P.falciparum TS-DHFR contains unique distant or nonactive regions that might modulate catalysis: (1) an N-terminal tail and (2) a linker region tethering DHFR to TS, and encoding a crossover helix that forms critical electrostatic interactions with the DHFR active site. The role of these nonactive sites in the bifunctional P. falciparum TS-DHFR is unknown. We report the first in-depth, pre-steady-state kinetic characterization of the full-length, wild-type (WT) P. falcipartan TS-DHFR enzyme and probe the role of distant, nonactive regions through mutational analysis. We show that the overall rate-limiting step in the WT P. falciparum TS-DHFR enzyme is TS catalysis. We further show that if TS is in an activated (liganded) conformation, the DHFR rate is 2-fold activated, from 60 s(-1) to 130 s(-1) in the WT enzyme. The TS rate is also reciprocally activated by similar to 1.5-fold if DHFR is in an activated, ligand-bound conformation. Mutations to the linker region affect neither catalytic rate nor domain-domain communication. Deletion of the N-terminal tail, although in a location remote from the active site, decreases the DHFR single rate and the bifunctional TS-DHFR rate by a factor of 2. The 2-fold activation of the DHFR rate by TS ligands remains intact, although even the activated N-terminal mutant has just half the DHFR activity of the WT enzyme. However, the reciprocal communication between TS active site and DHFR ligands is impaired in N-terminal mutants. Surprisingly, deletion of the analogous N-terminal tail in Leishmania major TS-DHFR causes a 3-fold enhancement of the DHFR rate from similar to 14 s(-1) to similar to 40 s(-1). In summary, our results demonstrate a complex interplay of domain-domain communication and nonactive-site modulation of catalysis in P. falciparum TS-DHFR. Furthermore, each parasitic TS-DHFR is activated by unique mechanisms, modulated by their nonactive site regions. Finally, our studies suggest the N-terminal tail of P. falciparum TS-DHFR is a highly selective, novel target for potential antifolate development in malaria.