- 學位論文
胃幽門螺旋桿菌尿嘧啶核甘酸激酶的表現質體建構以及功能分析
Molecular cloning and functional characterization of uridine monophosphate kinase from Helicobacter pylori
摘要
尿嘧啶核甘酸激酶(UMP kinases)在細菌中扮演重要且是個生存基本的酵素，它參與有關細菌內部三磷核酸的合成。被廣為人知的是它通常以六聚體的型態存在，而且由於它有很低的相似度和其他真核生物的單磷酸根合成酶，所以可以當成對抗菌類生長的抗生素研發目標。在本研究中，我們將胃幽門螺旋桿菌(菌株為26695)中的尿嘧啶核甘酸激酶利用基因重組的方法將它大量表現並純化。蛋白的分子量為27.562Kd,以及它的等電點為8,67. 重組的HP0777蛋白質在pH=8時有較低的溶解度(小於0.5ug /ul)，但是它的溶解度可藉由提高值到9來提高。尿嘧啶核甘酸激酶通常需要2mM的鎂離子來參與反應。尿嘧啶核甘酸激酶最好的反應溫度在30℃,如果將酵素放在65℃15分鐘後再測其活性在30℃的環境下，大概就只剩下10%的活性了。 重組蛋白HP0777的尿嘧啶核甘酸激酶活性被GTP所激活而被UTP所抑制。HP0777的酵素活性在GTP的濃度為0.15 mM時，酵素活型可被提高約15倍左右，相對於大腸桿菌在相關的文獻中提出GTP大概只能提高3~4倍左右。而UTP為1 mM在大約可以抑制70%左右的酵素活性。 存在錳離子2 mM的反應環境中相對於鎂離子的存在，重組蛋白HP0777的尿嘧啶核甘酸激酶活性大概為兩倍。而在存在鈷離子或是鎳離子的環境中相對於存在鎂離子的時候，活性分別大約為50%或7%。而存在鈣或銅離子時則無法測得酵素活性。而在測試何種pH值的實驗中，在存在鎂離子的情況下，在pH9有較高的活性，錳離子則是在pH7.4。 雖然HP0777的蛋白質序列相對於其它菌株的尿嘧啶核甘酸激酶大概有50%的同源性、70%的相似性。可是它卻有需要低濃度的GTP卻能大大提高其酵素活性以及存在錳離子的環境性有較高的酵素活性的兩大新發現。
並列摘要
Bacterial UMP kinases are important and essential enzymes which are involved in the multistep synthesis of nucleoside triphosphates. The known homohexamer from bacteria with no similar with eukaryotic organisms, might be a target for designing new antibacterial drugs. In this study, the HP0777 gene encoding UMP kinase in Helicobacter pylori strain 26695 was overexpressed. This protein has a molecular mass of 27.56 kD, and the Isoelectric point (pI) determination of 8.67. The recombinant HP0777 has low solubility at pH = 8 (≦ 0.5ug of protein/ul), but its solubility can be increased at an alkaline pH of 9. Rec-HP0777 showed UMP kinase activity in the presence of 2 mM MgCl2. The maximum UMP kinase activity of this protein appeared in reaction at 30 oC. About 10% enzyme activity remained after recombinant protein kept at 65 oC for 15 min and assayed at 30 oC. UMP kinase activity of rec-HP0777 protein was regulated by the activator GTP and inhibited by UTP. GTP at 0.15 mM could increase more than fifteen folds enzyme activity of rec-HP0777 protein, in contrast, 1 mM GTP increased 3-4 folds UMP kinase activity in E. coli from known published data. UTP at 1 mM decreased 70% of the UMP kinase activity for rec-HP0777 protein. UMP kinase activity of rec-HP0777 protein increased twice when magnesium was replaced with manganese at 2 mM. The activity reduced to 50%, 7% while cobalt or nickel ions were applied into reaction. When calcium or copper ions were substituted for manganese ions in the reaction mixture, the enzyme activity was not detectable. Determination of the enzyme activity at different pH value (6.5-9.5) indicated that the maximum UMP kinase activity of rec-HP0777 protein appeared in reaction buffer at pH 9.0 in present of magnesium and at pH 7.4 in present of manganese. Although the amino acid sequence of HP0777 shares almost 50% identity and 70% similarity to several known UMP kinase protein sequence from bacteria, the low concentration of GTP activator and manganese preference instead of magnesium for UTP kinase activity are novel discovery for HP0777 protein.