布魯菌半胱氨酸水解酶在甲硫氨酸循環(huán)中的催化活性研究
發(fā)布時間：2018-11-04 12:28
【摘要】：[目的]細菌的sah H基因編碼S-腺苷同型半胱氨酸水解酶(Sah H),該酶參與細菌的甲硫氨酸循環(huán)(AMC),調控細菌的多種生理功能。[方法]通過構建重組表達質粒p ET28a-Bru-sah H和p ET28a-Pse-sah H,分別表達布魯菌(Brucella abortus)S2308株和銅綠假單胞菌(Pseudomonas aeruginosa)PAO1株的Sah H重組蛋白Bru-Sah H和Pse-Sah H。將純化后的Bru-Sah H、Pse-Sah H以及我們前期表達純化的禽致病性大腸桿菌(avian pathogenic Escherichia coli,APEC)的Pfs和Lux S蛋白,分別在體外催化S-腺苷同型半胱氨酸(SAH),通過對產物同型半胱氨酸(HCY)的濃度測定,評價不同重組蛋白的催化活性,并對催化底物時產生的自誘導分子2(AI-2)活性進行檢測。[結果]Bru-Sah H和Pse-Sah H分別催化1 mmol·L-1SAH生成38和47μmol·L-1HCY,而APEC的Pfs和Lux S蛋白能催化相同濃度的SAH產生401μmol·L-1HCY。運用哈維弧菌BB170檢測上述底物的AI-2活性,結果表明只有同時采用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。[結論]Bru-Sah H能催化SAH生成HCY,為進一步研究sah H在布魯菌感染過程中的作用提供依據。
[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.
【作者單位】： 中國農業(yè)科學院上海獸醫(yī)研究所;揚州大學獸醫(yī)學院;奧本大學獸醫(yī)學院;
【基金】：國家自然科學基金項目(31572546,31370045) 國家農產品質量安全風險評估計劃項目(GJFP201700703)
【分類號】：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.
【作者單位】： 中國農業(yè)科學院上海獸醫(yī)研究所;揚州大學獸醫(yī)學院;奧本大學獸醫(yī)學院;
【基金】：國家自然科學基金項目(31572546,31370045) 國家農產品質量安全風險評估計劃項目(GJFP201700703)
【分類號】：S852.61
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