Nitroreductases are emerging as attractive bioremediation enzymes, with substrate promiscuity toward both natural and synthetic compounds. Recently, the nitroreductase NfnB from Sphingopyxis sp. strain HMH exhibited metabolic activity for dinitroaniline herbicides including butralin and pendimethalin, triggering the initial steps of their degradation and detoxification. However, the determinants of the specificity of NfnB for these herbicides are unknown. In this study, we performed structural and biochemical analyses of NfnB to decipher its substrate specificity. The homodimer NfnB is a member of the PnbA subgroup of the nitroreductase family. Each monomer displays a central α + β fold for the core domain, with a protruding middle region and an extended C-terminal region. The protruding middle region of Val75-Tyr129 represents a structural extension that is a common feature to members of the PnbA subgroup and functions as an opening wall connecting the coenzyme FMN-binding site to the surface, therefore serving as a substrate binding site. We performed mutational, kinetic, and structural analyses of mutant enzymes and found that Tyr88 in the middle region plays a pivotal role in substrate specificity by determining the dimensions of the wall opening. The mutation of Tyr88 to phenylalanine or alanine caused significant changes in substrate selectivity toward bulkier dinitroaniline herbicides such as oryzalin and isopropalin without compromising its activity. These results provide a framework to modify the substrate specificity of nitroreductase in the PnbA subgroup, which has been a challenging issue for its biotechnological and bioremediation applications.

中文翻译：

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NfnB 的结构和底物特异性决定因素，NfnB 是一种来自鞘氨醇菌的二硝基苯胺除草剂分解代谢硝基还原酶。应变 HMH。

硝基还原酶正在成为有吸引力的生物修复酶，其底物对天然和合成化合物都具有混杂性。最近，来自鞘氨醇菌的硝基还原酶 NfnB。菌株 HMH 对二硝基苯胺除草剂（包括布曲林和二甲戊灵）表现出代谢活性，触发了它们降解和解毒的初始步骤。然而，NfnB 对这些除草剂的特异性的决定因素是未知的。在这项研究中，我们对 NfnB 进行了结构和生化分析，以破译其底物特异性。同源二聚体 NfnB 是硝基还原酶家族的 PnbA 亚群的成员。每个单体都显示核心域的中央 α + β 折叠，具有突出的中间区域和延伸的 C 端区域。Val75-Tyr129 突出的中间区域代表结构延伸，它是 PnbA 亚群成员的共同特征，并作为将辅酶 FMN 结合位点连接到表面的开口壁，因此用作底物结合位点。我们对突变酶进行了突变、动力学和结构分析，发现中间区域的 Tyr88 通过确定壁开口的尺寸在底物特异性中起关键作用。Tyr88 突变为苯丙氨酸或丙氨酸导致对体积较大的二硝基苯胺除草剂（如安磺灵和异丙苯）的底物选择性发生显着变化，而不会影响其活性。这些结果提供了一个框架来修改 PnbA 亚组中硝基还原酶的底物特异性，