臺灣博碩士論文加值系統

細菌尿嘧啶雙磷酸葡萄醣去氫酶(UDP-glucose dehydrogenase, Ugd)的酵素活性受可逆的酪胺酸磷酸化的調控。經磷酸化的Ugd活性顯著提升，而經牛小腸鹼性去磷酸酶處理後活性下降。已知，大腸桿菌和克雷白氏肺炎桿菌CG43的酪胺酸激酶Wzc可藉由磷酸化Ugd來調控莢膜多醣體的生成。然而，受磷酸化調控的酪胺酸殘基尚未被確認。本研究藉由質譜儀的技術和序列比對，在克雷白氏肺炎桿菌Ugd所含的17個酪胺酸選了9個酪胺酸殘基，分別在10、91、150、210、217、242、249、265和335的位置，經點突變技術換成苯丙胺酸。再將這幾個定點突變後的蛋白質表現純化後，以磷酸化和去磷酸化的反應，分析其酵素活性。實驗結果發現Y91F和Y210F突變的酵素活性明顯高於野生型；尤其是Y91F突變株最為明顯。相反的，Y10F、Y242F和Y249F突變株測得的酵素活性遠比野生型的酵素低。我們也利用西方墨點法去確認了這些突變株是否有被磷酸化。但是，所有的突變Ugd被KpWzc磷酸化的程度都和野生型Ugd一樣好。由我們的結果可以得知Tyr10、Tyr242和Tyr249殘基對於Ugd酵素的活性影響非常大，而將酪胺酸置換成苯丙氨酸後的Ugd仍可被磷酸化。雖然，本研究結果並未確認被磷酸化調控的酪胺酸殘基，但是經由酵素動力學的實驗結果證實Tyr10、Tyr242和Tyr249殘基對於Ugd酵素活性的重要。

The enzymatic activity of bacterial UDP-glucose dehydrogenase (Ugd) has been shown to be under regulation by a reversible phosphorylation on the tyrosine residues. In Escherichia coli and Klebsiella pneumoniae CG43, the kinase Wzc is responsible for Ugd phosphorylation thereby modulates the synthesis of the polysaccharidic capsule. The phosphorylation of E. coli Ugd resulted in a significant increase of its activity whereas the treatment of Ugd with calf intestine alkaline phosphatase reduced its activity. However, the specific tyrosine residue subjected to the phosphorylation has not been identified. This study attempts to identify the critical tyrosine residues by site-directed mutagenesis of nine of the seventeen tyrosine residues contained in KpUgd, that were selected on the basis of their conserved nature and mass spectrum analysis. These single Tyr-to-Phe mutations of KpUgd were respectively at Tyr10, Tyr 91, Tyr150, Tyr210, Tyr217, Tyr242, Tyr249, Tyr265, and Tyr335. The mutant proteins were purified and their activities, either with or without incubation with KpWzc (Arg451-Lys722) were determined. The activities of UgdY91F and UgdY210F were higher than that of the wild-type Ugd. While UgdY10F, UgdY242F and UgdY249F exhibited a lower activity than that of the wild-type Ugd. We also used western blot hybridization to analyze if the mutations affected the tyrosine phosphorylation. The results indicated that all the Ugd mutants as well as the wild-type protein were able to be phosphorylated by the kinase KpWzc. Taken together, we have shown that the residue Tyr10, Tyr242 and Tyr249 were critical for the Ugd activity. In addition, the replacement of Tyr10, Tyr 91, Tyr150, Tyr210, Tyr217, Tyr242, Tyr249, Tyr265, and Tyr335 with Phe did not abolish the phosphorylation of Ugd. Although the specific tyrosine phosphorylation residue has not been identified, the enzymatic kinetic analysis of the mutant proteins demonstrated the critical role of the residue Tyr10, Tyr242 and Tyr249for Ugd activity.

Abstract in Chinese--------------------------------------Ⅰ
Abstract in English--------------------------------------Ⅱ
Acknowledgement------------------------------------------Ⅳ
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Abbreviations--------------------------------------------Ⅶ
Introduction----------------------------------------------1
Materials and Methods-------------------------------------7
Results---------------------------------------------------14
Discussion------------------------------------------------20
References------------------------------------------------24

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