結核分枝桿菌醣基轉移酵素之基因選殖與光親和性標記對酵素活性抑制研究

Abstract

結核分枝桿菌醣基轉移酵素之基因選殖與光親和性標記對酵素活性抑制研究

結核分枝桿菌（Mycobacterial cell)之霉菌酸阿拉伯半乳聚醣 (mycolyl-arabinogalactan, mAG)與脂阿拉伯甘露聚糖（lipoarabinomannan , LAM)

為其細胞壁之主要成分之一，其組成與分枝桿菌的存活性與致病性息息相關。LAM 與mAG之特殊組成其功能可保護分枝桿菌抵抗其宿主免疫系統的攻擊，並能構成 一道阻止抗生素入侵的屏障。因此若能深入地了解這些聚醣物質之生合成途徑， 將可進一步提供抗結核菌藥物的設計方向。最近，有數個參與在分枝桿菌細胞壁 多醣結構角色之醣基轉移酵素被鑑定出來，有些更可成功地在大腸桿菌的系統中 加以表達，但影響其酵素活性之主要氨基酸仍未被確認。

本研究計晝將針對結核菌細胞壁生合成途徑中之醣基轉移酵素如 galactofuranosyltransferase (GlfT1與GlfT2)進行酵素活性之主要氨基酸確認與抑 制之研究。首先、將針對GlfT1或GlfT2進行其基因之選殖與合成。隨後將利用 大腸桿菌進行蛋白質之表現，並搭配不同管柱層析法進行蛋白質的純化。這些純 化完之醣基轉移酵素將進行其生化特性之鑑定。另外，將合成具光親和性標記之 螢光雙醣類似物進行酵素活性抑制之研究。所獲得之光親和性標記-蛋白質複合 物將利用基質輔助雷射脫附游離飛行時間質譜或電喷灑離子化質譜確認被光親 和性標記所鍵結之氨基酸。

經由本計晝之執行，對於結核菌細胞壁生合成途徑中之醣基轉移酵素之結構 -活性之關係將可有進一步之了解，並對而所獲得之光親和性標記將可有機會作 為抗結核分枝桿菌藥物之應用。

Molecular Cloning and Photoaffinity Inhibitory Studies of Glycosyltransferases

from Mycobacterial tuberculosis

Mycolyl-arabinogalactan (mAG) and lipoarabinomannan (LAM) are major components in the mycobacterial cell wall essential for viability and virulence. The unusual components of mAG and LAM play important roles in protecting bacteria from the host immune system and providing a barrier for drug delivery. A better understanding of the biosynthetic pathway of glycan involved in the cell wall of mycobacteria will facilitate future design of anti-mycobacterial drugs. Recently, several glycosyltransferases (GTs) functioning on the assembly or the regulation of these polysaccharides scaffolds have been identified. Some of them were further over-expressed using the expression system. However, critical residues involved in the enzymatic activities have not been identified.

This proposal aims at gaining structure-activities relationships of galactofuranosyltransferases (GlfT1 and GlfT2) through critical residue identification and photoaffinity-labeled inhibitory studies. The GTs were thought to be essential in the assembly of carbohydrate building blocks and involved in the formation of mycobacterial cell wall. First, we will clone the GlfT1 and GlfT2 genes and overexpress the recombinant proteins in the E. coli strain. Subsequently, different chromatographic purifications will also be applied to isolate the expressed GTs from crude extract of an cell culture. The biological properties and the enzymatic activity of the GTs will be studied. In parallel, photoaffinity-labeled fluorescent disaccharide analogues will be synthesized and subjected to evaluate the inhibitory effect on GTs activities. The photoaffinity-labeled disaccharide analogues-GTs complexes will be digested with proteases and then subjected to matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) for active residue identification.

Thus, the execution of this project will advance not only the understanding of the critical role of various GTs in glycan biosynthetic pathway, but also the development of new type of photoaffinity tags for the pharmaceutical applications against TB infection.