Biochemical Structure and Function of Nitrate Reductase

9727982真核生物硝酸还原酶(NR；EC 1.6.6.1-3)的坎贝尔生物化学，在植物、藻类和真菌中催化NADH/NADPH催化硝酸盐还原为亚硝酸盐。研究人员对NR的结构和功能进行了研究，包括对其部分三维结构进行了建模。天然来源只提供少量的天然天然产物，为了提高对生物化学的了解，已经成功地使用了天然产物和活性片段的重组表达。本研究将在巴斯德毕赤酵母中重组表达具有活性的全反式硝酸还原酶，以生产获得完整三维结构所需的纯酶。NR的突变形式将被用来研究钼钼蝶呤辅助因子结合部位(Mo-MPT)、硝酸盐还原活性部位和亚基界面的功能。利用亚硫酸盐氧化酶的三维结构建立了天然橡胶的模型。通过光谱和稳态动力学分析研究天然橡胶的催化机理，通过合作完成稳态前动力学和EPR谱。现有的Mo-MPT配体突变体将在斯坦福同步辐射实验室通过X射线吸收光谱与野生型酶进行比较分析。这些研究将对NR的结构和功能提供一个完整而详细的图景，并指导未来的研究。硝酸还原酶是多中心氧化还原酶家族中一个独特的成员，这些酶催化长距离电子转移。硝态氮在农业中很重要，因为它是植物氮素积累的第一个酶促步骤。硝态氮在解决地下水和地表水硝酸盐污染引起的环境问题方面在生物技术方面也具有重要作用。NR是一种用于水分析的硝酸盐测试试剂盒的绿色催化剂，也是通过酶法反硝化消除饮用水中硝酸盐污染的系统的一部分。自然修复的基础研究正在为社会带来回报，因为它是环境生物技术的一种工具。

9727982 Campbell Biochemistry of eukaryotic nitrate reductase (NR; EC 1.6.6.1-3), catalyzing NADH/NADPH driven reduction of nitrate to nitrite in plants, algae and fungi, has been studied in this laboratory for 20 years. The investigator has studied structure and function of NR, including modeling part of its three-dimensional structure. Natural sources provide only small amounts of NR and to advance biochemical understanding, recombinant expression of NR and active fragments has been successfully used. The investigation will involve recombinant expression of active holo-NR in Pichia pastoris, a methylotrophic yeast, for producing purified enzyme needed to obtain complete 3-D structure. Mutant forms of NR will be used to study functionality in the Mo-molybdenum pterin cofactor binding site (Mo-MPT), nitrate reducing active site and subunit interface. A model of NR is being derived using the sulfite oxidase 3-D structure. Studies of NR's catalytic mechanism via spectral and steady-state kinetic analyses will be done with pre-steady state kinetics and EPR spectra done via collaboration. Existing mutants of ligands to Mo-MPT will be analyzed in comparison to wild-type enzyme via x-ray absorption spectroscopy at the Stanford Synchrotron Radiation Laboratory. These studies will yield a complete and detailed picture of NR structure and function and guide future studies. Nitrate reductase is a unique member of a multicenter redox enzyme family; these catalyze long-distance electron transfer. NR is important in agriculture since it serves as the first enzymatic step in nitrogen accumulation by plants. NR also has importance in biotechnology for addressing environmental problems arising from nitrate pollution of ground and surface waters. NR is a "green" catalyst for nitrate test kits for analysis of water and part of a system for eliminating nitrate pollution of potable water via enzymatic denitrification. Basic research on NR is paying off for society since it is a tool in environmental biotechnology.