Purification and characterization of nitrobenzene nitroreductase from Pseudomonas pseudoalcaligenes JS45
Purification and characterization of nitrobenzene nitroreductase from Pseudomonas pseudoalcaligenes JS45
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DOI：
10.1128/jb.177.13.3837-3842.1995
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发表时间：
1995-07
影响因子：
3.2
通讯作者：
Charles C. Somerville;S. Nishino;J. Spain
中科院分区：
文献类型：
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作者：
Charles C. Somerville;S. Nishino;J. Spain
假产碱假单胞菌JS 45以硝基苯作为唯一的碳、氮和能量来源生长。分解代谢途径包括还原为羟基氨基苯，然后重排为邻氨基苯酚和环分裂（S。F. Nishino和J.C.西班牙，应用环境Microbiol. 59：2520，1993）。通过硫酸铵沉淀、阴离子交换和凝胶过滤层析从JS 45的提取物中纯化硝基苯诱导的、氧不敏感的硝基还原酶。通过变性凝胶电泳检测到单个33-kDa多肽。通过凝胶过滤估计天然蛋白质的大小为30 kDa。该酶是一种黄素蛋白，具有紧密结合的黄素单酰肽辅因子，比例为每摩尔蛋白质2摩尔黄素。在初始NADPH浓度为0.5 mM时，硝基苯的Km为5 μ M。在初始硝基苯浓度为0.1 mM时，NADPH的Km为183 μ M。未检测到亚硝基苯作为硝基苯还原的中间体，但亚硝基苯是酶的底物，且亚硝基苯的比活性高于硝基苯。这些结果表明，亚硝基苯形成，但立即还原为羟基氨基苯。纯化的酶与硝基苯和NADPH孵育后，检测到的唯一产物是羟基氨基苯。羟基氨基苯不作为该酶进一步还原的底物。产物和中间体与母体化合物的两个双电子还原一致。此外，低Km值和酶合成的诱导控制表明硝基苯是该酶的生理底物。
Pseudomonas pseudoalcaligenes JS45 grows on nitrobenzene as a sole source of carbon, nitrogen, and energy. The catabolic pathway involves reduction to hydroxylaminobenzene followed by rearrangement to o-amino-phenol and ring fission (S. F. Nishino and J. C. Spain, Appl. Environ. Microbiol. 59:2520, 1993). A nitrobenzene-inducible, oxygen-insensitive nitroreductase was purified from extracts of JS45 by ammonium sulfate precipitation followed by anion-exchange and gel filtration chromatography. A single 33-kDa polypeptide was detected by denaturing gel electrophoresis. The size of the native protein was estimated to be 30 kDa by gel filtration. The enzyme is a flavoprotein with a tightly bound flavin mononucleotide cofactor in a ratio of 2 mol of flavin per mol of protein. The Km for nitrobenzene is 5 microM at an initial NADPH concentration of 0.5 mM. The Km for NADPH at an initial nitrobenzene concentration of 0.1 mM is 183 microM. Nitrosobenzene was not detected as an intermediate of nitrobenzene reduction, but nitrosobenzene is a substrate for the enzyme, and the specific activity for nitrosobenzene is higher than that for nitrobenzene. These results suggest that nitrosobenzene is formed but is immediately reduced to hydroxylaminobenzene. Hydroxylaminobenzene was the only product detected after incubation of the purified enzyme with nitrobenzene and NADPH. Hydroxylaminobenzene does not serve as a substrate for further reduction by this enzyme. The products and intermediates are consistent with two two-electron reductions of the parent compound. Furthermore, the low Km and the inducible control of enzyme synthesis suggest that nitrobenzene is the physiological substrate for this enzyme.