酵母菌 Pichia pastoris 中表現重組水稻蔗糖合成酶 RSuS3 之性質與結構探討

Abstract

蔗糖合成酶催化將蔗糖及UDP轉換為果糖及UDPG的可逆反應。水稻中發現三種RSus異構基因，RSus1及RSus2在水稻各組織中均有表現，而RSus3的表現具有穀實專一性。本論文以Pichia pastoris表現之重組RSuS3，探討其酵素性質與結構。 將建構於酵母菌P. pastoris中的RSus3 cDNA表現產物進行純化，加以分析。重組RSuS3僅在合成蔗糖方向具有較高的活性，分解蔗糖方向的活性極低。Mg2+可促進合成蔗糖方向的活性。醣中間代謝物對重組RSuS3活性的影響並不顯著，而磷脂質的存在與否對其活性也無明顯影響。 重組RSuS3蛋白質表面具有8個cysteine，並以電子顯微鏡觀察到原態蛋白質呈現四元體的形態。此外，已得到重組RSuS3蛋白質結晶及重原子共結晶，並收集原態蛋白質結晶之繞射數據，其解析度達3.53 Å，是蛋白質進行結晶繞射的初步成果。

Sucrose synthase catalyzes the reversible conversion of sucrose and UDP into fructose and UDPG. Among the three isogenes in rice (RSus1, RSus2, RSus3), the gene products of RSus1 and RSus2 are ubiquitously present in all rice tissues, while the expression of RSus3 is seed-specific. In this research, recombinant RSuS3 expressed from Pichia pastoris is used to study on the biological and structural characteristics of RSuS3. Expression plasmid containing RSus3 cDNA transformed into yeast P. pastoris for expression, and the recombinant product is purified to analyze its properties. The results show that the major activity of recombinant RSuS3 is to generate sucrose from fructose and UDPG, while the sucrose-cleavage activity of RSuS3 is very low. Besides, Mg2+ is able to promote the sucrose-synthesis activity of RSuS3. The presence of sugar metabolites have no significant effect on recombinant RSuS3 activity, neither do the presence of phospholipids. There are eight cysteines on the surface of recombinant RSuS3, and the native protein is present as a tetramer as revealed from the result of transmission electron microscopy (TEM). Furthermore, native crystal, heavy atom co-crystal and diffraction data of native crystal at 3.53 Å resolution has been obtained as the initial results of first step of X-ray crystallography.