胃幽門螺旋桿菌α1,3-岩藻醣轉移酶之晶體結構及催化機制

Abstract

胃幽門螺旋桿菌的alpha 1,3-岩藻醣轉移酶負責將岩藻醣從GDP-岩藻醣轉移到LacNAc雙醣上形成alpha 1,3-醣鍵，其反應產物為路易士X三醣。路易士X主要表現於胃幽門螺旋桿菌脂多醣類(LPS)的O-抗原，其結構類似於寄主的醣類抗原。此酵素的C端有一段七單元重複的白氨酸拉鍊結構幫助酵素以二聚體的四級結構來進行反應。在此論文研究中，我們成功地得到C端截去115個氨基酸的alpha 1,3-岩藻醣轉移酶晶體，並且解出酵素、酵素/受質(GDP-fucose)複合物、酵素/產物(GDP)複合物的三個晶體結構。此酵素之晶體結構包含兩個類似Rossmann折疊的區域，屬於典型的轉醣酶B族。GDP及GDP-岩藻醣都會專一地結合於酵素的活性區，同時造成酵素C端區域構形的改變。我們拿alpha 1,3-岩藻醣轉移酶結構與其他轉醣酶B族酵素的結構做比較、並且利用點突變的實驗證實，推測位於N端區域的第95號穀胺酸扮演催化時的通用鹼。將位於第195號位置的蛋白胺酸、第240號的天門冬胺酸、第249號的穀胺酸以及第250號的離胺酸點突變成丙胺酸後，發現這些突變種都會使酵素的活性明顯地降低。於alpha 1,3-岩藻醣轉移酶反應中加入EDTA並不影響其活性，表示此酵素並不需要二價的金屬離子參與反應。基於以上的觀察，我們提出一個alpha 1,3-岩藻醣轉移酶的催化機制。此外，從C端截去115個氨基酸的alpha 1,3-岩藻醣轉移酶在晶體中形成的二聚體特性也許能表達完整的酵素。

alpha 1,3-Fucosyltransferase (FucT) from H. pylori catalyzes the transfer of fucose from the donor GDP-beta-fucose in alpha 1,3-linkage to the acceptor beta-Gal-1,4-beta-GlcNAc (LacNAc) to produce the Lewis X trisaccharide. Lewis antigens are mainly expressed in the O-antigen of the H. pylori lipopolysaccharide and structurally similar to tumor-associated carbohydrate antigen found in the host. The enzyme contains a C-terminal heptad repeats that function as leucine zipper to facilitate the formation of a dimeric structure. In this thesis work, protein crystallization became successful only with the deletion of C-terminal 115 residues. We solved three crystal structures, including the protein, the protein-substrate (GDP-fucose) complex, and the protein-product (GDP) complex. The solved structures indicate that the enzyme is composed of two Rossmann-like fold domains, typical of the GT-B family of glycosyltransferases. Specific interactions with GDP and GDP-fucose bound to the active site induced conformational changes in the C-terminal domain. Structural comparison with other GT-B members suggested that Glu95 in the N-terminal domain plays the role of general base in catalysis, as confirmed by site-directed mutagenesis. Other mutants at Arg195, Asn240, Glu249 and Lys250 also showed significant decrease in the enzymatic activity. EDTA treatment showed that FucT does not require divalent metal ion. Based on these observations, a catalytic mechanism was proposed. Besides, the truncated FucT formed a dimer in crystal, which may characterize the full-length enzyme.