水稻蔗糖合成酶RSuS3突變株之分析與結構性質探討

Abstract

蔗糖合成酶催化蔗糖及UDP轉換為果糖與UDP-glucose的可逆反應。水稻中至少有六種蔗糖合成酶異構基因（RSus）。先前的研究已建構含有RSus3 cDNA的表現質體並轉形至酵母菌 P. pastoris 中表現。其表現之重組蛋白質在蔗糖合成方向的活性較蔗糖分解方向的活性明顯，與在大腸桿菌及酵母菌中表現的重組RSuS1不同。經核苷酸定序後發現，RSus3表現質體具有七個核苷酸突變，造成其表現之蛋白質具有四個胺基酸突變，分別是：T4P、V39A、D129G及F680S。在此將具有突變的重組蛋白質命名為mRSuS3。為探討這四個胺基酸突變對RSuS3活性的影響，本論文利用定位點突變法將突變逐一修復回正確的序列。活性分析的結果顯示，F680S-> F回復株在蔗糖合成與分解方向的活性都有顯著提升，但合成方向活性仍大於分解方向。其它三株回復株與mRSuS3在比活性上的差異則各不相同。根據RSuS3胺基酸序列分析和三級結構同源模擬的結果推論，Phe 680可能位於酵素的基質結合區域。 Lucifer yellow螢光標定的結果顯示，mRSuS3 四元體的36個cysteine當中，至少有六個cysteine暴露在mRSuS3蛋白質的表面。純化後的mRSuS3以2 % 醋酸鈾負染處理，可利用穿透式電子顯微鏡觀察到均勻的蛋白質粒子，直徑約15 nm。未來收集足夠的影像後可進行影像分類及3D影像重建。

Sucrose synthase catalyzes the reversible conversion of sucrose and UDP into fructose and UDP-glucose. There are at least six sucrose synthase genes in rice (RSus). In the previous study, an expression plasmid containing RSus3 cDNA was transformed into yeast Pichia pastoris for expression. The sucrose-synthesis activity of the expressed recombinant RSuS3 was more dominant than the sucrose-cleavage activity, which is different from the recombinant RSuS1 expressed in E. coli and in yeast. After nucleotide sequencing of the RSus3 expression plasmid, 7 nucleotide mutations were discovered, which led to four mutations, T4P, V39A, D129G and F680S, in amino acid sequence. Here the mutated recombinant protein is designated mRSuS3. In order to exam the effect of the four mutations on the activity of RSuS3, site-directed mutagenesis was employed in this research to revert the individual mutation. The result of activity assays showed that both sucrose-synthesis and sucrose-cleavage activities of F680S->F revertant was significantly higher than those of mRSuS3, but the sucrose-synthesis activity remained more dominant than sucrose-cleavage activity. The other three revertants showed differences in specific activity to various extent. According to amino acid sequence analysis and 3D structure homology modeling of RSuS3, Phe680 may be located in the substrate binding region of the enzyme. Among a total of 36 cysteines, there are at least 6 cysteines on the surface of mRSuS3 as revealed from the result of lucifer yellow labeling. Purified mRSuS3 was negatively stained with 2% uranyl acetate. The uniform particles with a diameter of about 15 nm were observed under transmission electron microscopy (TEM). Image classification and three dimensional reconstruction will be possible after sufficient images are collected.