Abstract：
Nitroreductases (NRs) are NAD(P)H-dependent flavoenzymes that reduce nitro aromatic compounds to their corresponding arylamines via the nitroso and hydroxylamine intermediates. Because of their broad substrate scope and versatility, NRs have found application in multiple fields such as biocatalysis, bioremediation, cell-imaging and prodrug activation. However, only a limited number of members of the broad NR superfamily (> 24 000 sequences) have been experimentally characterized. Within this group of enzymes, only few are capable of amine synthesis, which is a fundamental chemical transformation for the pharmaceutical, agricultural, and textile industries. Herein, we provide a comprehensive description of a recently discovered NR from Bacillus tequilensis, named BtNR. This enzyme has previously been demonstrated to have the capability to fully convert nitro aromatic and heterocyclic compounds to their respective primary amines. In this study, we determined its biochemical, kinetic and structural properties, including its apparent melting temperature (Tm) of 59 °C, broad pH activity range (from pH 3 to 10) and a notably low redox potential (-236 ± 1 mV) in comparison to other well-known NRs. We also determined its steady-state and pre-steady-state kinetic parameters, which are consistent with other NRs. Additionally, we elucidated the crystal structure of BtNR, which resembles the well-characterized Escherichia coli oxygen-insensitive NAD(P)H nitroreductase (NfsB), and investigated the substrate binding in its active site through docking and molecular dynamics studies with four nitro aromatic substrates. Guided by these structural analyses, we probed the functional roles of active site residues by site-directed mutagenesis. Our findings provide valuable insights into the biochemical and structural properties of BtNR, as well as its potential applications in biotechnology.
摘要：
硝基还原酶(NRs)是NAD(P)H依赖性黄素酶,其通过亚硝基和羟胺中间体将硝基芳族化合物还原为其相应的芳基胺。由于其广泛的底物范围和多功能性，NRs已在多个领域找到应用，如生物催化，生物修复，细胞成像和前药激活。然而,只有有限数量的广泛NR超家族成员(>24000序列)已经被实验表征。在这组酶中，只有少数人能够合成胺，这是制药的基本化学转变，农业,和纺织工业。在这里，我们提供了一个全面的描述最近发现的从芽孢杆菌的NR，名为BtNR。这种酶先前已被证明具有将硝基芳族和杂环化合物完全转化为它们各自的伯胺的能力。在这项研究中,我们确定了它的生化，动力学和结构特性，包括其59°C的表观熔化温度(Tm)，与其他众所周知的NRs相比，宽的pH活性范围（从pH3到10）和显着低的氧化还原电位（-236±1mV）。我们还确定了其稳态和预稳态动力学参数，这与其他NR是一致的。此外,我们阐明了BtNR的晶体结构，类似于特征明确的大肠杆菌氧不敏感的NAD（P）H硝基还原酶（NfsB），并通过与四种硝基芳族底物的对接和分子动力学研究，研究了其活性位点的底物结合。在这些结构分析的指导下，我们通过定点诱变研究了活性位点残基的功能作用。我们的发现为BtNR的生化和结构特性提供了有价值的见解，以及它在生物技术中的潜在应用。
