Biochemical Structure and Function of Nitrate Reductase

9727982坎贝尔在本实验室对真核硝酸还原酶（NR; EC 1.6.6.1-3）的生物化学进行了20年的研究，该酶催化植物、藻类和真菌中的NADH/NADPH驱动的硝酸盐还原为亚硝酸盐。 研究者对天然橡胶的结构和功能进行了研究，包括部分三维结构的建模。 天然来源仅提供少量NR，并且为了推进生物化学理解，NR和活性片段的重组表达已被成功使用。 研究将涉及在甲醇营养型酵母毕赤酵母中重组表达活性holo-NR，以生产获得完整三维结构所需的纯化酶。 NR的突变形式将用于研究钼-钼蝶呤辅因子结合位点（Mo-MPT）、硝酸盐还原活性位点和亚基界面的功能。NR的模型是使用亚硫酸盐氧化酶的三维结构。 通过光谱和稳态动力学分析研究NR的催化机理将与通过合作完成的前稳态动力学和EPR光谱一起完成。 现有的突变体的配体，以钼-MPT将进行分析相比，野生型酶通过X-射线吸收光谱在斯坦福大学同步辐射实验室。 这些研究将产生一个完整的和详细的图片NR的结构和功能，并指导未来的研究。 硝酸还原酶是多中心氧化还原酶家族的独特成员;这些催化长距离电子转移。 NR在农业中很重要，因为它是植物氮积累的第一个酶促步骤。 NR在生物技术中对于解决地下和地表沃茨的硝酸盐污染引起的环境问题也具有重要意义。 NR是一种“绿色”催化剂，用于硝酸盐检测试剂盒，用于分析水和通过酶促脱氮消除饮用水硝酸盐污染的系统的一部分。NR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.