Somerville C C, Nishino S F, Spain J C
Armstrong Laboratory, Tyndall Air Force Base, Florida 32403-5323, USA.
J Bacteriol. 1995 Jul;177(13):3837-42. doi: 10.1128/jb.177.13.3837-3842.1995.
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.
假产碱假单胞菌JS45能够以硝基苯作为唯一的碳、氮和能量来源进行生长。其分解代谢途径包括将硝基苯还原为羟基氨基苯，随后重排为邻氨基苯酚并发生环裂解（S.F. Nishino和J.C. Spain，《应用与环境微生物学》59:2520，1993）。通过硫酸铵沉淀，随后进行阴离子交换和凝胶过滤色谱，从JS45提取物中纯化出一种硝基苯诱导型、对氧气不敏感的硝基还原酶。变性凝胶电泳检测到一条单一的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，每摩尔蛋白质中紧密结合2摩尔黄素单核苷酸辅因子。在初始NADPH浓度为0.5 mM时，硝基苯的Km值为5 μM。在初始硝基苯浓度为0.1 mM时，NADPH的Km值为183 μM。未检测到亚硝基苯作为硝基苯还原的中间体，但亚硝基苯是该酶的底物，且其对亚硝基苯的比活性高于对硝基苯的比活性。这些结果表明亚硝基苯已形成，但会立即被还原为羟基氨基苯。将纯化的酶与硝基苯和NADPH一起孵育后，仅检测到羟基氨基苯作为产物。羟基氨基苯不能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次双电子还原一致。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。
