幽門螺旋菌formamidase之結構-功能性分析及CrdSR雙分子系統參與一氧化氮逆境之調控機轉

The structure-function relationship of formamidase and CrdSR-associated regulator in responding to nitric oxidative stress in Helicobacter pylori

洪秋蓮(Chiu-Lien Hung)

指導教授 ： 王雯靜

國立清華大學/生命科學院/分子與細胞生物研究所/博士(2009年)

摘要

幽門螺旋菌（Helicobacter pylori）是一種感染人類胃部的格蘭氏陰性菌，可抵擋胃部強烈胃酸的環境，並藉由金屬離子體內平衡以幫助細菌群聚與確立至胃上皮黏膜細胞上，進而使細菌侵入到細胞內。本論文主要以結構-功能關聯性研究formamidase 及雙分子訊息傳遞系統。第一部份為 formamidase：AmiF （EC 3.5.1.49）可催化短鏈的 amides 產生 carboxylic acid 及氨，使得幽門螺旋菌具有抗酸的能力。依據 AmiF 序列比對分析其屬於nitrilase superfamily 其中一員，nitrilase superfamily酵素可以水解非胜肽的碳-氮鍵，如水解nitriles產生其相關的carboxylic acids並釋放出氨，這個反應為nitriles的去毒性作用，以作為防禦性的化學物質，且可以利用於生物復育之領域。為了了解幽門螺旋菌 AmiF在 nitrilase superfamily 演化上的意義，我們選殖 H. pylori amiF 基因並測定其結構。AmiF在不對稱單元為為 homodimer型式存在，每個單體呈現 α-β-β-α 三明治摺疊結構，與 nitrilase superfamily 蛋白質結構摺疊方式類似。AmiF 活性催化殘基為 Cys166、Glu60 及 Lys133，形成一個具有C-E-K catalytic triad，且在 nitrilase superfamily 中皆具高度保留性。並且完成 AmiF 活性中心經突變後 apo 及 ligand 形式之晶體結構，比較apo及liganded之結構與功能之分析，並與D-NCAase 進行比較，以瞭解此nitrilase superfamily催化活性機制及篩選受質特異性探討。 第二部份為研究Two-component system雙分子系統(TCSs) CrdSR 調控胃幽門螺旋菌適應NO刺激之機制。幽門螺旋菌的感染與許多胃部疾病有關連（如胃潰瘍、慢性胃炎等）。TCSs為細菌感受外在環境的改變進而調控基因的表現以適應外在環境改變的系統之一。本論文的研究目的是為了瞭解 TCSs 是否參與抵抗宿主免疫反應，結合系統生物學、生物資訊、以及分生方法來探討細菌如何利用TCSs進行訊息傳遞。使用 TFBSFinder 和 magiic-tfbs2 預測 response regulator (RR) 是如何辨認 DNA-binding 序列進而進行基因調控；並於化學藥劑一氧化氮壓力中使用細胞存活率測定的方法，證明CrdSR對於幽門螺旋菌適應一氧化氮壓力的確扮演重要角色。這些結果將有助於我們瞭解幽門螺旋菌在逆境的適應與生存模式。綜合實驗結果顯示：幽門螺旋菌受到一氧化氮壓力時，CrdSR雙分子系統藉由保守性 AAACNC 辯識序列進行相關基因調控，幽門螺旋菌可以藉由雙分子系統與金屬依賴性的訊息傳遞路徑及DNA修補重組基因交互作用適應宿主免疫系統攻擊。

關鍵字

幽門螺旋菌 ； 雙分子系統

並列摘要

Helicobacter pylori colonizes the human gastric mucosa and causes severe gastric disease. In the hostile ecological niche, maintaining proper metal ion metabolism is of critical importance for the pathogen. This investigation focuses on the structure-function study of two factors: formamidase and copper resistance determinant-associated regulators (CrdSR) in two component systems (TCSs). The first part is formamidase AmiF that hydrolyzes formamide to produce formic acid and ammonia belongs to the branch 2 of nitrilase superfamily. Nitrile-degrading enzymes are valuable in the synthesis of pharmaceuticals and commodity chemicals as well as in potential bioremediation applications. These enzymes are members of the carbon-nitrogen hydrolase, or called nitrilase, superfamily which catalyze the hydrolysis of a wide range of non-peptide carbon-nitrogen bonds. The nitrilase superfamily consists of 13 branches with a conservative C-E-K catalytic triad. The crystal structure of AmiF was solved to 1.75 Å resolution using single-wavelength anomalous dispersion methods. The structure consists of a homohexamer related by 3-fold symmetry in which each subunit has an aa four-layer architecture characteristic of the nitrilase superfamily. One exterior a layer faces the solvent, whereas the other one associates with that of the neighbor subunit, forming a tight aaaa dimer. The apo and liganded crystal structures of an inactive mutant C166S were also determined to 2.50 and 2.30 Å, respectively. These structures reveal a small formamide-binding pocket that includes C166, E60, and K133 catalytic residues, in which C166 acts as a nucleophile. Analysis of the liganded AmiF and N-carbamoyl D-amino acid amidohydrolase binding pockets reveals a common Cys-Glu-Lys triad, another conserved glutamate, and different subsets of ligand-binding residues.
Helicobacter pylori is a gastric pathogen that infects approximately half of the human population. Persistent infection of this peculiar microbe causes chronic inflammation, which may progressively lead to severe forms of gastrointestinal diseases. Two component systems (TCSs) are the primary type of the signal perception system to respond the extracellular stimuli by informing the cellular transcriptional machinery in prokaryotes and lower eukaryotes. In an effort to understand how H. pylori responds to immune attack, we found that a histidine kinase crdS was upregulated upon nitric oxide challenge. This study proposes to tackle this important signaling system in prokaryotes using a combined systematic biology, bioinformatics and molecular biology approach. Transcription levels of potential genes that might be regulated by CrdSR were analyzed by quantitative real-time RT-PCR, that elucidate TFBSFinder and magiic-tfbs2 could be achievable to predict regulon. Furthermore, viability assay of H. pylori survival in NO from the chemical agent demonstrated that CrdSR and its regulon are important to responding the NO stress. We propose that characterization of CrdR DNA binding consensus with tandem or mirror repeat of AAACNC and many CrdR-regulated genes encode metal-efflux genes, DNA repair, recombination, outer membrane proteins, amino acid metabolism and energy production thus confirming the previously proposed role of CrdSR system in regulation of metal-efflux functionality. We proposed that CrdSR is involved in above mentioned important functions. These results will increase our understanding of how H. pylori responds to different environmental cues as well as the knowledge of bacterial signaling flow in a changing environment. Taken together, these results indicated that the TCS that governs the metal-dependent pathway is engaged in responding to NO stress. These results will increase our understanding of how H. pylori responds to different environmental cues as well as the knowledge of bacterial signaling flow in the context of a changing environment.

並列關鍵字

Helicobacter pylori ； nitrilase superfamily ； two-component systems

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