腸炎弧菌磷脂醯絲胺酸合成酶基因及生化特性之研究

Abstract

腸炎弧菌在台灣是主要引起食物中毒的病原菌之一，磷脂醯絲胺酸合成酶 (Pss) 為弧菌合成磷脂質組成份的一個決定性的酵素，可催化胞苷二磷酸二醯甘油和L型絲胺酸成為磷脂醯絲胺酸及5’-胞核苷單磷酸鹽。本研究欲探討腸炎弧菌中磷脂醯絲胺酸合成酶基因 (pss) 在細菌中的表現及此基因所生成的蛋白質之功能及特性。pss的mRNA於生長的指數期表現，其轉錄起始點位於轉譯起始點上游37個核苷酸，並藉由報導基因的試驗及RNA聚合酶之電泳位移分析證明pss基因的啓動子區域。另外，建構可能的調控者VPA0041之缺陷株發現VPA0041並不能調控pss基因在轉錄層次的表現。此外，由免疫沉澱法及液相串連式質譜儀證明腸炎弧菌的細胞內確實會轉譯出Pss蛋白質。為證實Pss蛋白質具有催化的功能，利用大腸桿菌大量表現可溶的重組Pss，並將其純化進行酵素活性分析。藉由基質輔助雷射解析串聯飛行時間質譜儀，可以證明Pss酵素催化基質後的產物磷脂醯絲胺酸的生成，使用分光光譜儀可以證實Pss酵素催化反應之產物5’-胞核苷單磷酸鹽的產生。另外，Pss最適反應溫度為40℃，最適pH為8，並受到20 mM二價金屬離子的抑制。再者，經由建構Pss的蛋白質立體結構模型，以及將模型與基質進行分子對接的模擬，及定點突變的製作，推測其酵素活性區存在於與其他磷脂酶D家族具保守性的兩個HKD (His(X)Lys(X)4Asp) 區域。弧菌屬中的Pss是具高保守性的蛋白質，本論文研究結果可促進了解其他弧菌之磷脂醯絲胺酸合成酶的基因及生化特性。

Vibrio parahaemolyticus is one of the main food pathogens that cause foodborne illness in Taiwan. Phosphatidylserine synthase (Pss) is a crital enzyme to synthesize phospholipid, which can catzlyze cytidine diphosphate diacylglycerol and L‐serine to form phosphatidylserine and cytidine 5’‐ monophosphate. This study would investigate the gene expression of the phosphatidylserine synthase gene (pss) in V. parahaemolyticus, and the function and the characteristics of Pss. pss mRNA was found to be expressed mainly during the exponential phase. The transcriptional start site of pss was mapped through sequencing and was identified at −37 nucleotides upstream of the start codon. In addition, the promoter was identified using a lux reporter assay and gel shift assay with an RNA polymerase. Furthermore, one predicted Pss regulator deletion mutant ΔVPA0041 was constructed, however, VPA0041 was found that it was no effect on regulating the transcriptional level of pss. To analyze the catalytic activity, a soluble form of His6‐tagged recombinant Pss was overexpressed and purified from Escherichia coli. Using matrix‐assisted laser desorption ionizationtime of flight mass spectrometry, the formation of phosphatidylserine was proved. The production of cytidine 5’‐monophosphate was confirmed by using spectrophotometer. Moreover, the optimum temperature of Pss was 40°C and the optimum pH of Pss was 8. The activity of Pss was inhibited by 20 mM divalent metal. In addition, through the construction of the three-dimensional structure of Pss, the simulation of molecular docking of the model with the substrate, and site-directed mutagenesis, the active region of the enzyme is presumed to be the two HKD (His(X)Lys(X)4Asp) regions conserved with other phospholipase D families. Since Pss is a conserved protein in Vibrio, this study can help to understand genetic and biochemical characterization of Pss in other Vibrio.