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染料脱色过氧化物酶(DyP-type过氧化物酶)是含有亚铁血红素,能降解各种有毒染料的一类蛋白.为了研究运动发酵单胞菌Zymomonas mobilis ZM4 (ATCC 31821)中一种新的DyP-type过氧化物酶的特点和功能,以Z.mobilis基因组DNA为模板,通过PCR扩增目的基因,克隆到大肠杆菌表达载体pET-21b(+)中.通过ZmDyP与其他DyP-type过氧化物酶的比对,发现它们存在着共同保守氨基酸D149、R239、T254、F256和GXXDG结构基序,说明ZmDyP是Dyp-type过氧化物酶家族的一个新成员.经IPTG诱导大肠杆菌中pET21 b(+)-ZmDyP表达,并将表达的酶进行金属螯合层析纯化.SDS-PAGE分析表明,纯酶分子量为36 kDa,而活性染色显示分子量为108 kDa,表明该酶在活性状态下可能是一个三聚体.光谱扫描显示ZmDyP有一个典型的亚铁血红素吸收峰,说明它是含有亚铁血红素的蛋白.对ZmDyP性质进行了研究,发现以2,2-二氨-双(3-乙基苯并噻唑-6-磺酸)ABTS为底物,ZmDyP表现出更高的转化效率.这些研究结果丰富了DyP-type 过氧化物酶家族信息,并且为ZmDyP的结构功能和反应机制研究奠定了基础. 相似文献
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Paolo Ascenzi Elisabetta De Marinis Chiara Ciaccio Massimo Coletta 《Biochemical and biophysical research communications》2009,380(2):392-31
During infection, Mycobacterium leprae is faced with the host macrophagic environment limiting the growth of the bacilli. However, (pseudo-)enzymatic detoxification systems, including truncated hemoglobin O (Ml-trHbO), could allow this mycobacterium to persist in vivo. Here, kinetics of peroxynitrite (ONOOH/ONOO−) detoxification by ferryl Ml-trHbO (Ml-trHbOFe(IV)O), obtained by treatment with H2O2, is reported. Values of the second-order rate constant for peroxynitrite detoxification by Ml-trHbOFe(IV)O (i.e., of Ml-trHbOFe(III) formation; kon), at pH 7.2 and 22.0 °C, are 1.5 × 104 M−1 s−1, and 2.2 × 104 M−1 s−1, in the absence of and presence of physiological levels of CO2 (∼1.2 × 10−3 M), respectively. Values of kon increase on decreasing pH with a pKa value of 6.7, this suggests that ONOOH reacts preferentially with Ml-trHbOFe(IV)O. In turn, peroxynitrite acts as an antioxidant of Ml-trHbOFe(IV)O, which could be responsible for the oxidative damage of the mycobacterium. As a whole, Ml-trHbO can undertake within the same cycle H2O2 and peroxynitrite detoxification. 相似文献
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Ascenzi P De Marinis E Coletta M Visca P 《Biochemical and biophysical research communications》2008,373(2):197-201
Kinetics of ferric Mycobacterium leprae truncated hemoglobin O (trHbOFe(III)) oxidation by H2O2 and of trHbOFe(IV)O reduction by NO and NO2− are reported. The value of the second-order rate constant for H2O2-mediated oxidation of trHbOFe(III) is 2.4 × 103 M−1 s−1. The value of the second-order rate constant for NO-mediated reduction of trHbOFe(IV)O is 7.8 × 106 M−1 s−1. The value of the first-order rate constant for trHbOFe(III)ONO decay to the resting form trHbOFe(III) is 2.1 × 101 s−1. The value of the second-order rate constant for NO2−-mediated reduction of trHbOFe(IV)O is 3.1 × 103 M−1 s−1. As a whole, trHbOFe(IV)O, generated upon reaction with H2O2, catalyzes NO reduction to NO2−. In turn, NO and NO2− act as antioxidants of trHbOFe(IV)O, which could be responsible for the oxidative damage of the mycobacterium. Therefore, Mycobacterium leprae trHbO could be involved in both H2O2 and NO scavenging, protecting from nitrosative and oxidative stress, and sustaining mycobacterial respiration. 相似文献
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