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  • 學位論文

水稻蔗糖合成酶催化反應機制與蛋白質結構之探討

Studies on the catalytic mechanism and the structure of rice sucrose synthase

指導教授 : 王愛玉

摘要


蔗糖合成酶 (SuS) 催化蔗糖與 UDP 轉化為果糖與 UDP-glucose 的可逆反應。水稻中蔗糖合成酶 (RSuS) 至少由七種異構基因 (RSus) 所表現,且不同異構酶間的胺基酸序列高度相似。 本研究的目標是解析 RSuS 結構與功能間的關係。基質結合分析的結果顯示,RSuS 在蔗糖分解方向的先導基質為 UDP,而在蔗糖合成方向的先導基質為果糖。嘗試利用小角度 X-ray 散射、蛋白質結晶繞射及同源模擬的方式,進行 RSuS 蛋白質結構的解析。利用阿拉伯芥蔗糖合成酶 AtSuS1 為模版進行同源模擬,得到接近全長的 RSuS 模擬結構。由模擬結構的資訊推測,Gln307 及 E-X7-E motif 與基質結合相關。將 Gln307 突變成 Asn 或 Ser 會導致 RSuS1 酵素活性喪失,RSuS1(Q307N) 仍保有與果糖的結合能力,但 RSuS1(Q307S) 則喪失與果糖結合的能力。在糖基轉移酶 3、4 及 5 家族中,E-X7-E motif 是保守性高的區塊。此區塊中的突變株雖不影響 RSuS3 蛋白質四元體的形成,但僅 F680Y 與E686D 保有部分活性,E678D、E678Q、F680S 及 E686Q 則無酵素活性。對此區塊內無活性的突變株進行基質結合能力分析,結果顯示 E678D、E678Q、F680S 及 E686Q 皆保有與 UDP 結合的能力,但都無法與果糖結合。對 RSuS3 與保有活性的突變株 F680Y 及 E686D 進行酵素動力學分析,結果顯示這兩個胺基酸突變不僅影響酵素與 UDP-glucose 的結合能力,也影響酵素活性的展現。

並列摘要


Sucrose synthase (SuS, EC 2.4.1.13) catalyzes the reversible conversion of sucrose and UDP into fructose and UDP-glucose. The enzyme is encoded by at least seven RSus genes in rice. The amino acid sequences of different RSuS are highly similar. The objective of this study is to elucidate the structure-function relationships of RSuS. The results of substrate binding assay shown that UDP and fructose are the leading substrate of sucrose cleavage and sucrose synthesis direction, respectively. Different approaches including small angle X-ray scattering, protein crystallography and homology modeling, have been attempted to obtain the structure information of RSuS. The theoretical structure of partial RSuS, which was built with the structure of Arabidopsis sucrose synthase 1 as template, revealed that Gln307 and E-X7-E motif of RSuS was involved in fructose binding. Changing the Gln307 to Asn or Ser resulted in completely loss of enzyme activity. Substrate binding assays revealed that the mutant RSuS1(Q307N) retained the capability of fructose binding while RSuS1(Q307S) did not. The E-X7-E motif is conserved in members of glycosyltransferase family 4 (GT4) and other two GT families. The results of enzyme activity assay showed that the mutants E686D and F680Y retained partial enzyme activity and the mutants E678D, E678Q, F680S and E686Q were inactive; however, the tetrameric conformation was maintained in these mutant proteins. The results of substrate binding assays revealed that the mutants E6786D, E686Q, F680S and E686Q could bind UDP but lost the capability of sucrose binding and fructose binding. The kinetic parameters of RSuS3, F680Y and E686D indicated that the residues in E-X7-E motif are important in UDP-glucose binding and enzyme activity.

參考文獻


Tsai, C.Y. and Wang, A.Y. (2003) Identification of rice manganese-dependent protein kinases that phosphorylate sucrose synthase at multiple serine residues. Bot Bull Acad Sin 44: 141-150.
陳姿利 (2005) 水稻蔗糖合成酶 RSuS2 在酵母菌 Pichia pastoris 中表現及生化性質檢定. 國立臺灣大學微生物與生化學研究所.
黃玉嬌 (2006) 水稻蔗糖合成酶 RSuS1 野生型與突變型蛋白質之表現與檢定. 國立臺灣大學微生物與生化學研究所.
蔡逸君 (2006) 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. 國立臺灣大學微生物與生化學研究所.
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