"大豆內切-1,4-β-甘露聚醣酶之受質專一性與 轉醣基能力的結構觀點與探討"

Abstract

內切-1,4-β-甘露聚醣酶 (endo-1,4-β-mannanase, β-mannanase, EC. 3.2.1.78) 在植物發芽和生長調控上扮演重要的角色，此酵素主要功能為水解β-1,4甘露聚醣使植物細胞壁軟化，以利於種子胚軸之突出與胚乳的消耗，然而目前對於植物來源β-mannanase 之結構及功能研究甚少。而本論文選擇大豆之β-mannanase作為研究對象，原因除了大豆是全球重要經濟作物外，在食品、飼料和工業應用上亦有很大的潛力。經基因體探勘，大豆中有21個β-mannanase基因，其中GmMAN19-1於親緣關係樹上較有其獨特性，因此以此基因為首要目標。 即時PCR結果顯示GmMAN19-1 基因於發芽7天後之子葉組織中表現，而經純化後的GmMAN19-1重組蛋白為一嗜酸性酵素且在pH 4.6有最大活性，且對直鏈型多醣有較好之水解能力。為了獲得更詳細的資訊，我們利用蛋白質結晶學解出GmMAN19-1以及其與受質五醣之複合體結構，意外發現此複合體結構包含兩種五醣的結合模式，分別呈現出糖苷水解酶受質 (subsites：-3,-2,-1,+1,+2) 和轉糖酵素受質 (subsite：-5,-4,-3,-2,-1) 的結合狀態。此外，GmMAN19-1與其他真菌來源之β-mannanase的結構比較顯示，GmMAN19-1於結構上多出了兩個延伸的loop，造成了較狹窄的活性位裂口。以解出的複合體結構為基礎，為嘗試提高GmMAN19-1對支鏈性甘露聚醣的選擇性，進行循理設計將GmMAN19-1突變。在我們所構築的五個突變株中，以Q267W最具潛力，因其對於支鏈型甘露聚醣的關華豆膠相對於野生株有50% 的比活性提升，而Q267W突變株，還有Y264W突變株及其複合物等蛋白質結構，也被進行結構解析並探討。整體而言，我們的研究結果提供GmMAN19-1受質專一性與轉醣基能力的結構觀點，並顯示了植物型β-mannanase和真菌來源之β-mannanase於結構上的差異。且特別是，Q267W對於支鏈型受質的水解有相當的潛力，提供了未來對於GmMAN19-1進行酵素工程以及大豆分子育種的依據。

Endo-1,4-β-mannanase (β-mannanase, EC. 3.2.1.78) is a hydrloase that catalyzes cleavage of β-1-4 bonds in the mannan polymer. This enzyme family is involved in soften of the mannan-rich cell walls and consumption of endosperm, which is benifical to radicle protrusion upon seed germination. However, there is limited information about the structural and functional relationship of plant-type β-mannanase. In this study, plant-type β-mannanases from soybean (Glycine max) were studied, since soybean is not only a globally important commercial crops, but also a potential material for use in food, feed or industrial applications. Using genome mining, we find out that there are 21 types of β-mannanase gene in the genome of soybean, and GmMAN19-1 was selected as primiary target due to its unique position on phylogentic tree. RT-PCR data showed GmMAN19-1 was expressed only in the cotyledons tissue after 7-day germination. Purified recombinant GmMAN19-1 was acidophilic with a pH optimum of 4.6, and exhibited a higher activity to linear polysaccharides. For detailed information, crystal sturctures of GmMAN19-1 in apo form and in complex with mannopentose were determined. Intriguingly, the complex structure existed two distinct binding modes of mannopentaose, presented as the substrates for glycohydrolase (subsides -3, -2, -1, +1, +2) and transglycohydrolase (subsides -5, -4, -3, -2, -1), respectively. In addition, structural comparison of GmMAN19-1 with other β-mannanases from fungus reveals that GmMAN19-1 has two extended loops, producing a narrower active site cleft. Based on the solved structure of GmMAN19-1/pentaose complex, rational design was conducted to engineer GmMAN19-1 in an attempt to alter the substrate selectivity toward branched mannans. Among the 5 mutants we constructed, the most promising Q267W showed a 50% increase in specific activity toward the branched-mannan guar gum by comparison with the wild-type enzyme. GmMAN19-1-Q267W, GmMAN19-1-Y264W and its complex were also structurally characterized. Taken together, our findings provide structural insights into the substrate specificity and transglycosylation activity of GmMAN19-1 and demonstrate the structural differences between plant-type and fungal β-mannanase. In particular, Q267W mutant shows potential to hydrolysis branched substrate, which providing a basis for further enzymatic engineering of GmMAN19-1 and molecular breeding of soybean.