Zheng Chunli, Zhang Yanfei, Liu Yuandong, Wu Anna, Xia Lexian, Zeng Jia, Liu Jianshe, Qiu Guanzhou
School of Resource Processing and Bioengineering, Central South University, Changsha 410083, People's Republic of China.
Curr Microbiol. 2009 Jun;58(6):586-92. doi: 10.1007/s00284-009-9375-1. Epub 2009 Feb 19.
Adenosine 5'-phosphosulfate (APS) reductase is a key enzyme involved in the pathways of sulfate reduction and sulfide oxidation in the biological sulfur cycle. In this study, the gene of APS reductase from Acidithiobacillus ferrooxidans was cloned and expressed in Escherichia coli, the soluble protein was purified by one-step affinity chromatography to apparent homogeneity. The molecular mass of the recombinant APS reductase was determined to be 28 kDa using SDS-PAGE. According to optical and EPR spectra results of the recombinant protein confirmed that the iron-sulfur cluster inserted into the active site of the protein. Site-directed mutation for the enzyme revealed that Cys110, Cys111, Cys193, and Cys196 were in ligation with the iron-sulfur cluster. The [Fe4S4] cluster could be assembled in vitro, and exhibited electron transport and redox catalysis properties. As we know so far, this is the first report of expression in E. coli of APS reductase from A. ferrooxidans.
腺苷-5'-磷酸硫酸还原酶（APS还原酶）是生物硫循环中参与硫酸盐还原和硫化物氧化途径的关键酶。在本研究中，嗜酸氧化亚铁硫杆菌的APS还原酶基因被克隆并在大肠杆菌中表达，通过一步亲和层析将可溶性蛋白纯化至表观均一。使用SDS-PAGE测定重组APS还原酶的分子量为28 kDa。根据重组蛋白的光学和电子顺磁共振光谱结果证实，铁硫簇插入到该蛋白的活性位点。对该酶进行定点突变表明，半胱氨酸110、半胱氨酸111、半胱氨酸193和半胱氨酸196与铁硫簇相连。[Fe4S4]簇能够在体外组装，并表现出电子传递和氧化还原催化特性。据我们目前所知，这是嗜酸氧化亚铁硫杆菌的APS还原酶在大肠杆菌中表达的首次报道。
