Institute of Biochemistry and Biology, Department of Molecular Enzymology , University of Potsdam , 14476 Potsdam , Germany.
Institute for Experimental Physics , Free University of Berlin , Arnimallee 14 , 14195 Berlin , Germany.
Biochemistry. 2018 May 15;57(19):2889-2901. doi: 10.1021/acs.biochem.8b00128. Epub 2018 Apr 30.
The Mo/Cu-dependent CO dehydrogenase (CODH) from Oligotropha carboxidovorans is an enzyme that is able to catalyze both the oxidation of CO to CO and the oxidation of H to protons and electrons. Despite the close to atomic resolution structure (1.1 Å), significant uncertainties have remained with regard to the reaction mechanism of substrate oxidation at the unique Mo/Cu center, as well as the nature of intermediates formed during the catalytic cycle. So far, the investigation of the role of amino acids at the active site was hampered by the lack of a suitable expression system that allowed for detailed site-directed mutagenesis studies at the active site. Here, we report on the establishment of a functional heterologous expression system of O. carboxidovorans CODH in Escherichia coli. We characterize the purified enzyme in detail by a combination of kinetic and spectroscopic studies and show that it was purified in a form with characteristics comparable to those of the native enzyme purified from O. carboxidovorans. With this expression system in hand, we were for the first time able to generate active-site variants of this enzyme. Our work presents the basis for more detailed studies of the reaction mechanism for CO and H oxidation of Mo/Cu-dependent CODHs in the future.
寡养单胞菌的钼/铜依赖型一氧化碳脱氢酶（CODH）是一种能够同时催化 CO 氧化为 CO 和 H 氧化为质子和电子的酶。尽管其结构接近原子分辨率（1.1Å），但对于独特的钼/铜中心的底物氧化反应机制以及催化循环中形成的中间体的性质，仍存在很大的不确定性。到目前为止，由于缺乏合适的表达系统，无法在活性位点进行详细的定点突变研究，因此对活性位点氨基酸的作用的研究受到了阻碍。在这里，我们报告了在大肠杆菌中功能性异源表达寡养单胞菌 CODH 的建立。我们通过动力学和光谱学研究的结合，详细地表征了纯化酶，并表明它是以与从寡养单胞菌中纯化的天然酶相当的形式进行纯化的。有了这个表达系统，我们第一次能够生成该酶的活性位点变体。我们的工作为未来更详细地研究钼/铜依赖型 CODH 的 CO 和 H 氧化反应机制奠定了基础。
