Although nitrobenzene and its derivatives(NBDs)are important industrial materials,they can cause severe dam-age to the environment.Physical,chemical and biological methods are currently used to degrade residual NBDs in the envi-ronment,among which the biological method is considered to be the most promising,given its advantages such as low cost, little secondary pollution,and ecological restoration. 4-chloronitrobenzene(4,CNB),one of the highly toxic and dangerous NBDs,can cause persistent physiological damage to human beings via direct or indirect contact.The 2-amino-5-chloromuconic semialdehyde dehydrogenase(CnbD)from Comamonas testosteroni CNB1 is an important component in-volved in 4,CNB degradation.The crystal structure of CnbD can help us to understand the mechanism of this enzyme and make full use of CnbD to degrade 4,CNB in the environment.ThecnbD gene was cloned into the prokaryotic expression vector pET-28,a(+)and the CnbD protein was overexpressed in Escherichia coli.The CnbD protein was purified with Ni2+-chelating affinity chromatography and gel filtration chromatography.Protein crystals were cultivated at 289,K and diffracted to resolution of 0.18,nm and 0.22,nm respectively at the wavelength of 0.10,nm in SSRF.With the molecular-replacement (MR)method,the three-dimensional structure of the protein was successfully determined.%硝基苯类化合物作为一种重要的化工原料,在工业广泛应用的同时也产生了环境污染.目前降解硝基苯类污染物的方法有物理法、化学法和生物法,其中生物修复法具有低成本、无二次污染、生态恢复性好等优点.硝基苯类化合物中的对氯硝基苯是一种高毒、危险的合成品,会对人体造成直接或间接的生理损伤.来源于睾丸酮丛毛单胞菌CNB1(Comamonas testosteroni CNB1)的2-氨基-5-氯粘康酸半醛脱氢酶(2-amino-5-chloromuconic semialdehyde de-hydrogenase,CnbD)是对氯硝基苯降解通路中的关键酶.研究CnbD的晶体结构有助于完善生物修复治理对氯硝基苯污染的机制.实验以睾丸酮丛毛单胞菌CNB1中的CnbD为研究对象,将基因cnbD克隆到pET-28,a(+)上进行原核表达.通过镍离子亲和层析和凝胶过滤层析等纯化方法获得重组蛋白CnbD.在289,K下获取了2种重组蛋白CnbD晶体,并在同步辐射光源BL19,U光束线的0.10,nm波长X射线衍射下得到最高衍射分辨率分别为0.18,nm和0.22,nm的衍射数据.采用分子置换法,初步解析了CnbD的同源四聚体结构.
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