枯草桿菌UDP-glucosyltransferase中Phe240胺基酸殘基對檞黃素醣基化之影響

Abstract

檞黃素為植物體中常見的物質，更是人類飲食中被大量攝取的營養素，是一種對人體有益的物質。檞黃素所具有的抗氧化、抗發炎、預防心血管疾病與抗癌等生理功效，在最近幾年更是吸引許多學者投入研究。檞黃素醣基化合物 (quercetin glycoside)是植物體主要的儲存形式，其醣基化的位向更是與人類食用後的吸收與利用有關聯。 枯草桿菌的BcGT-1酵素是一種醣基轉移酶 (glycosyltransferase)，能使UDP-glucose上的葡萄糖轉移至檞黃素結構上的4個氫氧基。本篇研究將BcGT-1酵素Phe-240施以定點突變，發現：經由改變胺基酸特性不僅有機會得到單一位向單醣基檞黃素，也可能對酵素活性造成影響。 由圓二色光譜分析各Phe-240突變酵素顯示，單一點突變BcGT-1酵素中Phe-240，並不會對酵素結構產生大改變，但卻影響酵素反應性與受質醣基化的位向，我們推測Phe-240在酵素活性區內扮演與醣基接受者作用之角色，此外應尚有其他殘基，如Phe-132及Phe-138，亦與檞黃素有重要的作用關係。進一步的酵素殘基與受質之相對關係仍有賴於蛋白質結構的解析。

Quercetin, a common constituent in the diet, is claimed to exert beneficial health effects. In addition to its anti-inflammatory and antioxidant activities, quercetin also exerts cardiovascular disease prevention effect and anti-cancer effect. In plants and most plant-derived foods, quercetin is largely present as glycosylated forms (quercetin glycoside). The nature of glycosylation is known to influence the efficiency of quercetin absorption and biological function. The glycosyltransferase from Bacillus cereus (BcGT-1) is a member of the UGTs, which is capable of transferring glucose into four different hydroxyl groups of quercetin. In this study we demonstrate site-directed mutagenesis on acceptor binding site. Mutation of Phe240 drastically changed the regioselectivity of quercetin glycosylation and catalytical activity. CD inspection revealed that the secondary structures of all studied Phe240 mutants did not significantly perturbed. Yet, the enzyme activity and product specificity were varied. We confirm Phe240 is a significant residue that mediates the orientation of sugar acceptor and consequently control, at least partially, the enzymatic activity and product specificity. Other residues, such as Phe132 and Phe138, may also involve in the acceptor binding. More detailed topology of BcGT-1 can be addressed when the high resolution protein structure is available.