- 學位論文
結核分支桿菌之dehydroquinate synthase與受質複合體晶體結構研究
Crystal Structure of the Dehydroquinate Synthase Substrate Complex from Mycobacterium tuberculosis
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摘要
3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (DAHPS) 及dehydroquinate synthase (DHQS) 為 shikimate pathway 中的第一步及第二步驟，皆為抗微生物、抗寄生蟲及除草劑的可能標鈸。DAHPS 催化 phosphoenolpyruvate 及 erythrose 4-phosphate 縮合成 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP)；DHQS 需要 NAD，將DAHP 轉換成 3-dehydroquinate (DHQ)。本研究的主要目的為決定 DAHPS 及 DHQS 的 X 光三度空間結構，以找出可能的抑制藥物。我們培養Helicobacter pylori DAHPS (HpDAHPS) 晶體，並利用微晶體篩選 (microseeding screening)、添加物篩選、除水法 (dehydration)、cryoprotectant 測試，增進 HpDAHPS 的散射圖譜。Mycobacterium tuberculosis DHQS (MtDHQS) 或加上 DAHP，分別產生解析度為2.07和3.00 Å的晶體。N domain 由 Rossmann fold 所組成；C domain 含多個 α-helix 結構。DAHP 位在兩個 domain 之間。不含 DAHP 的 MtDHQS – 不含配位體 (ligand) 及僅含 NAD+ 的 MtDHQS，形成 open form；反之含 DAHP 的 MtDHQS 轉型成 closed form。比較真核及原核生物的 DHQS 顯示與 DAHP 結合的殘基高度保留，提供未來結構上的抑制物開發。以IC50 測試找出 RH00573 的 IC50 為64.88 µM。
並列摘要
3-Deoxy-D-arabino-heptulosonate 7-phosphate synthase (DAHPS) and dehydroquinate synthase (DHQS) are enzymes that catalyze the first and second step of the shikimate pathway. Since the shikimate pathway are absent in mammals, they are potential targets for new antimicrobial agents, anti-parasitic agents and herbicides. DAHPS catalyzes the condensation of phosphoenolpyruvate with erythrose 4-phosphate to give 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP)；DHQS is a NAD-dependent enzyme that converts DAHP into 3-dehydroquinate (DHQ). In this study, we investigated the crystal structure of Helicobacter pylori DAHPS (HpDAHPS) and Mycobacterium tuberculosis DHQS (MtDHQS) as the first step for drug design. The purified HpDAHPS was crystallized and improved diffraction pattern via microseeding screening, additive screening, dehydration and cryprotectant test. Crystallization of MtDHQS, fllowing addition DAHP, gave crystals with 2.07 and 3.00 Å resolution respectively. N-terminal domain has the Rossmann-type architecture, and C-terminal domain contains multipleα-helix structure. MtDHQS in the absence of DAHP, either unliganed or in the presence of NAD+, was an open form；in contrast, DAHP binding turned the MtDHQS into closed form. Structures’ comparison reveals the DAHP binding residues are highly conserved between prokaryotes and eukaryote, which provided a basis for the future structure-guided design of DHQS inhibitors. By using drug IC50 measurement, we try to find out possible inhibitors. The IC50 of drug RH00573 is 64.88 µM.
參考文獻
(1994). The CCP4 suite: programs for protein crystallography. Acta Crystallogr D Biol Crystallogr 50, 760-763.
Arcuri, H.A., Borges, J.C., Fonseca, I.O., Pereira, J.H., Neto, J.R., Basso, L.A., Santos, D.S., and de Azevedo, W.F., Jr. (2008). Structural studies of shikimate 5-dehydrogenase from Mycobacterium tuberculosis. Proteins 72, 720-730.
Bender, S.L., Mehdi, S., and Knowles, J.R. (1989). Dehydroquinate synthase: the role of divalent metal cations and of nicotinamide adenine dinucleotide in catalysis. Biochemistry 28, 7555-7560.
Blaser, M.J. (1992). Helicobacter pylori: its role in disease. Clin Infect Dis 15, 386-391.
Carpenter, E.P., Hawkins, A.R., Frost, J.W., and Brown, K.A. (1998). Structure of dehydroquinate synthase reveals an active site capable of multistep catalysis. Nature 394, 299-302.