水稻蔗糖合成酶RSuS1 野生型與突變型蛋白質之表現與檢定

Abstract

蔗糖合成酶催化蔗糖與UDP 轉化為果糖與UDPG 的可逆反應。水稻中此酵素由至少六種RSus 基因所表現。本論文研究的對象為RSus1 基因表現產物 RSuS1，利用大腸桿菌表現重組RSuS1 進行結構與功能的分析。 RSuS1 經由序列分析已知為 GT-B 摺疊構型的糖基轉移酶，且結構與 glycogen synthase 相似。重組 RSuS1 突變株的初步分析發現Asp303、Gln307、Tyr310、His463 及His502 等氨基酸可能在基質結合或者活性區中扮演重要角色；而Tyr310、His463 及Arg94 等胺基酸則為維持四級結構所必需。以大腸桿菌表現之重組RSuS1，其分解與合成方向活性受到二價金屬離子不同的影響。鎂、鈣、錳及鈷離子會促進重組RSuS1 蔗糖合成方向的活性，但抑制重組 RSuS1 蔗糖分解方向的活性。醣代謝之中間代謝產物對 RSuS1 活性沒有顯著的影響。Triton X-114 partition 結果發現重組 RSuS1 不是integral 形式的膜蛋白質，phospholipids 對活性也無顯著影響。

Sucrose synthase (SuS) catalyzes the reversible conversion of sucrose and UDP into fructose and UDPG. The enzyme is encoded by at least six RSus genes in rice. In this study, recombinant RSuS1 expressed and purified from E. coli was used to study on the structure – function relationship of the enzyme. The results of sequence analysis and 3D structure modeling suggest that the RSuS1 is a GT-B fold glycosyltransferase of the GT4 family in CAZy classification. Mutants analysis show that the Asp303, Gln307, His463 and His502 play important roles in substrate binding or active sit. Tyr310, His463 and Arg94 are important in tetrameric structure. Divalent cations, Mg2+, Mn2+, Ca2+, and Co2+ stimulate the recombinant RSuS1 synthetic activity, but inhibit the cleavage activity. Metabolites do not affect the recombinant RSuS1 activity significantly. Triton X-114 partition analysis shows that recombinant RSuS1 is not an integral membrane protein. The phospholipids do not affect the RSuS1 activity.