酵母菌 Pichia pastoris 中表現之重組水稻蔗糖合成酶 RSuS1 及 RSuS3 之性質探討

Characterization of Recombinant Rice Sucrose Synthase RSuS1 and RSuS3 Expressed in Pichia pastoris

蔡青霖(Ching-Lin Tsai)

指導教授 ： 王愛玉

國立臺灣大學/生命科學院/微生物與生化學研究所/碩士(2007年)

https://doi.org/10.6342/NTU.2007.10580

摘要

蔗糖合成酶催化蔗糖及 UDP 轉換為果糖及 UDPG 的可逆反應。在目前已知的六種水稻蔗糖合成酶異構基因 (RSus) 中，RSus1 及 RSus3 已被轉殖至酵母菌 Pichia pastoris 並表現重組 RSuS 蛋白質。然而核苷酸定序結果顯示位於 P. pastoris 表現質體中的 RSus1 及 RSus3 cDNA 皆帶有突變。本研究建構帶有正確 RSus1 及 RSus3 ORF 序列之表現質體，並成功以 P. pastoris 表現重組野生型 RSuS1 及 RSuS3 蛋白質，同時兩重組蛋白質皆以 DEAE-Sephacel 離子交換層析、 Sephacryl S-300 膠體過濾層析及 Resource-Q 陰離子交換快速液相層析進行純化。
重組野生型 RSuS3 蛋白質的蔗糖分解與合成方向比活性皆比突變型 RSuS3 蛋白質 (mRSuS3) 高出許多，同時兩者間蔗糖分解與合成方向比活性之比值也有明顯差異。由酵素動力學的分析結果，比較對 UDPG 的親和力，重組野生型 RSuS3遠高於重組 mRSuS3。這些分析結果顯示位於 mRSuS3 上的變異，包括 T4P、V39A、D129G 及 F680S，在 RSuS3 與 UDPG 的結合上，以及 RSuS3 在蔗糖合成及分解方向的活性影響上，皆扮演著重要的角色。
由酵素動力學的研究結果亦發現，重組 RSuS1 對 UDPG 及果糖的親和力遠高於 mRSuS1；在蔗糖合成方向比活性的比較上，重組 RSuS1 亦高於 mRSuS1。重組 RSuS1 與 mRSuS1 之 C 端序列的差異可能為影響活性及與基質結合的重要原因。

關鍵字

水稻蔗糖合成酶

並列摘要

Sucrose synthase (SuS) catalyzes the reversible conversion of sucrose and UDP into fructose and UDP-glucose. Among six known rice Sus genes (RSus), RSus1 and RSus3 were previously transformed into yeast Pichia pastoris and the recombinant RSuS proteins were expressed successfully. However, the results of nucleotide sequencing of expression plasmids containing RSuS1 or RSuS3 cDNA showed that there were mutations in both RSus1 and RSus3. In this research, the expression plasmids carrying the wild-type open reading frame of RSus1 and RSus3 were constructed and transformed into P. pastoris for expression. Both recombinant proteins were purified by DEAE-Sephacel ion-exchange chromatography, Sephacryl S-300 gel filtration chromatography and Resource-Q FPLC system.
The specific activity of recombinant RSuS3 in both sucrose cleavage and synthesis direction were much higher than those of mutated RSuS3 (mRSuS3), and the ratio of sucrose-cleavage to sucrose-synthesis specific activity was also different from mRSuS3. Moreover, the binding affinity for UDPG of RSuS3 was much higher than that of mRSuS3, as revealed by the kinetic analysis data. These results suggested that the mutations, T4P, V39A, D129G and F680S, in mRSuS3 may affect the substrate binding and also the catalytic activity in both sucrose cleavage and synthesis directions.
The kinetic analysis data also showed that the binding affinity of both UDPG and fructose of RSuS1 are much higher than those of mRSuS1. The specific activity of recombinant RSuS1 in sucrose synthesis direction is much higher than that of mRSuS1. The results indicated that the mutations in the C terminus of mRSuS1 may affect the binding of UDPG and fructose to the enzyme and its activity.

並列關鍵字

RSuS

參考文獻

Tsai ZC, Wang AY (2003) Identification of rice manganese-dependent protein kinases that phosphorylate sucrose synthase at multiple serine residues. Botanical bulletin of Academia Sinica 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. 碩士論文. 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. 碩士論文. 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. 碩士論文. 國立臺灣大學微生物與生化學研究所

黃卓萱 (2007) 水稻蔗糖合成酶 RSuS3 突變株之分析與結構性質探討. 碩士論文. 國立臺灣大學微生物與生化學研究所