水稻蔗糖合成酶中 E-X7-E motif 之功能探討

Abstract

摘要 蔗糖合成酶 (SuS) 在植物中催化蔗糖和 UDP 轉換為果糖和 UDPG 的可逆轉換。此酵素屬於糖基轉移酶中第四家族 (GT4)。GT4家族的成員在序列上有著一段具高度保守性的序列：E-X7-E motif，此序列兩端為 Glu，中間間隔七個保守性較低的胺基酸。除了 GT4 外，GT3 及 GT5 家族的糖基轉移酶也具有此序列。已有研究發現，E-X7-E motif對於這些酵素的活性相當重要，推測其功能可能為穩定基質，並使基質正確定位至催化活性區。 本研究探討此段序列在水稻蔗糖合成酶 RSuS3 催化活性上的可能功能。以定位點突變法建構四個RSuS3突變株 (E678D, E678Q, E686D, E686Q)，並利用酵母菌 Pichia pastoris 表現系統進行重組野生型與突變蛋白質的表現與純化。由活性測定結果顯示，RSuS3(E686D) 突變株仍保有部分活性，其對蔗糖與 UDP 之 Km 值明顯增大。由本研究結果推測，這 E-X7-E motif 上的兩個 Glu 對於基質以及過渡態的穩定扮演重要角色，這兩個位點的突變可能會干擾基質與酵素的相互作用，造成 Km 值的增大或是酵素活性的喪失，

Sucrose synthase catalyzes the reversible conversion of sucrose and UDP into fructose and UDPG in plants. The enzyme belongs to the glycosyltransferase GT4 family. The members of GT4 have a highly conserved motif, the E-X7-E motif, in their sequence. This motif has two glutamates at two sides, which are separated by seven less conserved amino acids. Besides GT4, enzymes in families GT3 and GT5 also contain this motif. It has been reported that E-X7-E motif is important in the activity of enzyme, it may have the function in substrate stabilization, and position substrates into active site. This work examined the possible function of this motif in rice sucrose synthase 3 (RSuS3). Site-directed mutagenesis was employed to construct four mutants of RSuS3 (E678D, E678Q, E686D, E686Q) and the recombinant wild-type and mutant proteins were expressed and purified from Pichia pastoris. The results of activity assays showed that only RSuS3(E686D) retained partial enzyme activity. The Km values of mutant RSuS3(E686D) were significantly increased. The results of this study suggested that the two glutamates in the E-X7-E motif may have an important role in substrate binding, and also in the stabilization of transition state; mutagenesis on these two residues may disrupt the interaction of enzyme and substrates, which resulted in the increase of Km or the loss of enzyme activity.