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Towards a complete structure-function description of the denitrification pathway

实现反硝化途径的完整结构功能描述

基本信息

批准号：
BB/D016290/2
负责人：
Samar Hasnain
金额：
$ 26.55万
依托单位：
University of Liverpool
依托单位国家：
英国
项目类别：
Research Grant
财政年份：
2008
资助国家：
英国
起止时间：
2008 至无数据
项目状态：
已结题

来源：
https://gtr.ukri.org/projects?ref=BB%2FD016290%2F2
关键词：
Towards complete structure function description

项目摘要

The denitrification process is a four-step reduction of nitrate to dinitrogen via the intermediates nitrite (NO2) and the gaseous N-oxides NO and N2O. The loss of fixed nitrogen via these steps to inert dinitrogen is of major importance to the terrestrial and oceanic nitrogen cycles and has agronomic, environmental, and medical impacts. The current proposal builds on our recent exciting results on nitrite reductases from two dentrifiers, A. xylosoxidans and A. cycloclastes, and nitrous oxide reducatse (N2OR) from A. cycloclastes (our unpublished results). These enzymes catalyse the first committed step, namely the reduction of NO2, and the last step of the denitrification process. The project is aimed towards reaching a 'complete' understanding of the important steps of denitrification catalysed by these enzymes through combined structural, and enzymological studies and site directed mutations of these enzymes. Thus, for example, in the case of NiR, we propose to test different aspects of our proposed catalytic mechanism (PNAS, 23 Aug 2005) experimentally. Since the enzyme used in these studies was the green AcNiR, we wish to test the general occurrence of these intermediates in CuNiR turnover by extending this work to the blue AxNiR, for which we have some 20 potentially relevant mutant NiRs, many of which have been structurally characterized in their resting states. In the case of N2OR, on the basis of two structures we have obtained very recently, we have proposed a reaction mechanism which requires confirmation through an extensive experimental structure-function programme. Even though the recent structure determinations have advanced our knowledge in terms of the mechanism, they have also highlighted challenges in understanding how the difficult chemistry of N2O reduction is achieved by the novel CuZ cluster.

反硝化过程是通过中间体亚硝酸盐 (NO2) 和气态氮氧化物 NO 和 N2O 将硝酸盐还原为二氮的四步过程。通过这些步骤将固定氮损失为惰性二氮对于陆地和海洋氮循环具有重要意义，并且具有农艺、环境和医学影响。当前的提案建立在我们最近对来自两种齿菌（A. xylosoxydans 和 A. cycloclastes）的亚硝酸还原酶以及来自 A. cycloclastes 的一氧化二氮还原酶（N2OR）的令人兴奋的结果（我们未发表的结果）的基础上。这些酶催化第一个关键步骤，即 NO2 的还原，以及反硝化过程的最后一步。该项目旨在通过结合结构和酶学研究以及这些酶的定点突变，对这些酶催化的反硝化的重要步骤达成“完整”的了解。因此，例如，就 NiR 而言，我们建议通过实验测试我们提出的催化机制的不同方面（PNAS，2005 年 8 月 23 日）。由于这些研究中使用的酶是绿色 AcNiR，我们希望通过将这项工作扩展到蓝色 AxNiR 来测试这些中间体在 CuNiR 周转中的普遍发生情况，为此我们有大约 20 个潜在相关的突变体 NiR，其中许多突变体已在静止状态下进行了结构表征。就 N2OR 而言，基于我们最近获得的两种结构，我们提出了一种反应机制，需要通过广泛的实验结构功能程序进行确认。尽管最近的结构测定提高了我们在机制方面的知识，但它们也凸显了理解新型 CuZ 团簇如何实现 N2O 还原的困难化学过程的挑战。