布鲁菌半胱氨酸水解酶在甲硫氨酸循环中的催化活性研究
发布时间：2018-11-04 12:28
【摘要】：[目的]细菌的sah H基因编码S-腺苷同型半胱氨酸水解酶(Sah H),该酶参与细菌的甲硫氨酸循环(AMC),调控细菌的多种生理功能。[方法]通过构建重组表达质粒p ET28a-Bru-sah H和p ET28a-Pse-sah H,分别表达布鲁菌(Brucella abortus)S2308株和铜绿假单胞菌(Pseudomonas aeruginosa)PAO1株的Sah H重组蛋白Bru-Sah H和Pse-Sah H。将纯化后的Bru-Sah H、Pse-Sah H以及我们前期表达纯化的禽致病性大肠杆菌(avian pathogenic Escherichia coli,APEC)的Pfs和Lux S蛋白,分别在体外催化S-腺苷同型半胱氨酸(SAH),通过对产物同型半胱氨酸(HCY)的浓度测定,评价不同重组蛋白的催化活性,并对催化底物时产生的自诱导分子2(AI-2)活性进行检测。[结果]Bru-Sah H和Pse-Sah H分别催化1 mmol·L-1SAH生成38和47μmol·L-1HCY,而APEC的Pfs和Lux S蛋白能催化相同浓度的SAH产生401μmol·L-1HCY。运用哈维弧菌BB170检测上述底物的AI-2活性,结果表明只有同时采用AEPC的Pfs和Lux S蛋白催化SAH,才能形成有活性的AI-2分子,而Bru-Sah H和Pce-Sah H均不能催化SAH形成活性AI-2分子。[结论]Bru-Sah H能催化SAH生成HCY,为进一步研究sah H在布鲁菌感染过程中的作用提供依据。
[Abstract]:[objective] the sah H gene of bacteria encodes S-adenosine homocysteine hydrolase (Sah H), which participates in the regulation of bacterial physiological functions by methionine cycle (AMC),. [methods] Recombinant expression plasmids p ET28a-Bru-sah H and p ET28a-Pse-sah H were constructed to express Sah H recombinant proteins Bru-Sah H and Pse-Sah H of Brucella (Brucella abortus) S2308 strain and Pseudomonas aeruginosa (Pseudomonas aeruginosa) PAO1 strain, respectively. The Pfs and Lux S proteins of purified Bru-Sah Hapse-Sah H and the purified avian pathogenic Escherichia coli (avian pathogenic Escherichia coli,APEC were used to catalyze S- adenosine homocysteine (SAH), in vitro, respectively. The catalytic activity of different recombinant proteins was evaluated by measuring the concentration of homocysteine (HCY), and the self-induced molecular 2 (AI-2) activity of the substrate was determined. [results] Bru-Sah H and Pse-Sah H catalyzed 1 mmol L-1SAH to produce 38 and 47 渭 mol L -1 HCY, respectively, while APEC Pfs and Lux S protein could catalyze the production of 401 渭 mol L -1 HCY at the same concentration of SAH. The AI-2 activity of the above substrates was determined by BB170 of Vibrio Harvey. The results showed that only the Pfs and Lux S proteins of AEPC could catalyze SAH, to form active AI-2 molecules. Neither Bru-Sah H nor Pce-Sah H can catalyze SAH to form active AI-2 molecules. [conclusion] Bru-Sah H can catalyze the production of HCY, from SAH and provide evidence for further study on the role of sah H in brucellosis infection.
【作者单位】： 中国农业科学院上海兽医研究所;扬州大学兽医学院;奥本大学兽医学院;
【基金】：国家自然科学基金项目(31572546,31370045) 国家农产品质量安全风险评估计划项目(GJFP201700703)
【分类号】：S852.61
[Abstract]:[objective] the sah H gene of bacteria encodes S-adenosine homocysteine hydrolase (Sah H), which participates in the regulation of bacterial physiological functions by methionine cycle (AMC),. [methods] Recombinant expression plasmids p ET28a-Bru-sah H and p ET28a-Pse-sah H were constructed to express Sah H recombinant proteins Bru-Sah H and Pse-Sah H of Brucella (Brucella abortus) S2308 strain and Pseudomonas aeruginosa (Pseudomonas aeruginosa) PAO1 strain, respectively. The Pfs and Lux S proteins of purified Bru-Sah Hapse-Sah H and the purified avian pathogenic Escherichia coli (avian pathogenic Escherichia coli,APEC were used to catalyze S- adenosine homocysteine (SAH), in vitro, respectively. The catalytic activity of different recombinant proteins was evaluated by measuring the concentration of homocysteine (HCY), and the self-induced molecular 2 (AI-2) activity of the substrate was determined. [results] Bru-Sah H and Pse-Sah H catalyzed 1 mmol L-1SAH to produce 38 and 47 渭 mol L -1 HCY, respectively, while APEC Pfs and Lux S protein could catalyze the production of 401 渭 mol L -1 HCY at the same concentration of SAH. The AI-2 activity of the above substrates was determined by BB170 of Vibrio Harvey. The results showed that only the Pfs and Lux S proteins of AEPC could catalyze SAH, to form active AI-2 molecules. Neither Bru-Sah H nor Pce-Sah H can catalyze SAH to form active AI-2 molecules. [conclusion] Bru-Sah H can catalyze the production of HCY, from SAH and provide evidence for further study on the role of sah H in brucellosis infection.
【作者单位】： 中国农业科学院上海兽医研究所;扬州大学兽医学院;奥本大学兽医学院;
【基金】：国家自然科学基金项目(31572546,31370045) 国家农产品质量安全风险评估计划项目(GJFP201700703)
【分类号】：S852.61
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