Optimized inhibition assays reveal different inhibitory responses of hydroxylamine oxidoreductases from beta- and gamma-proteobacterial ammonium-oxidizing bacteria
Optimized inhibition assays reveal different inhibitory responses of hydroxylamine oxidoreductases from beta- and gamma-proteobacterial ammonium-oxidizing bacteria
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DOI：
10.1016/j.bbrc.2016.05.041
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发表时间：
2016-07-29
影响因子：
3.1
通讯作者：
Yamazaki, Toshimasa
中科院分区：
文献类型：
--
作者：
Nishigaya, Yuki;Fujimoto, Zui;Yamazaki, Toshimasa
氨氧化细菌（AOB）是一种普遍存在的化学自养细菌，以羟胺为能量源，将氨（NH3）转化为亚硝酸盐（NO2-）。农田大量施用铵肥促进了AOB的过度生长，导致氮淋溶和氧化亚氮气体排放。为了抑制这些不利现象，硝化抑制剂、AOB特异性杀菌剂被广泛应用于施肥农田。然而，由于目前使用的抑制剂的毒性和弱效应，需要新的硝化抑制剂。为了开发针对AOB中硝化作用关键酶羟胺氧化还原酶（hydroxylamine oxidoreductase， HAO）的新型硝化抑制剂，我们建立了包括简化的HAO纯化程序和对纯化酶和AOB活细胞进行高通量HAO活性测定的抑制剂评价体系。新的检测系统使我们能够观察到β -变形菌AOB （β AOB）、欧洲亚硝基单胞菌（NeHAO）和γ -变形菌AOB （γ AOB）海洋亚硝基球菌（NoHAO）的HAOs对苯肼（NeHAO的一种众所周知的自杀抑制剂）的不同抑制反应。与此一致的是，N. europaea、亚硝化单胞菌sp. JPCCT2和MOB的多形亚硝化螺旋菌的活细胞对苯肼的反应高于γ AOB N. oceani。我们的同源性模型研究表明，在这两类细菌中，由于两个残基的取代，MOB和γ AOB的不同抑制反应源自于HAOs底物结合位点周围的不同局部环境。本文报道的结果强烈建议筛选针对β AOB的NeHAO和γ AOB的NoHAO的抑制剂，以开发具有广泛抗AOB谱的针对hao的硝化抑制剂。(C) 2016年由Elsevier Inc.出版。
Ammonia-oxidizing bacteria (AOB), ubiquitous chemoautotrophic bacteria, convert ammonia (NH3) to nitrite (NO2-) via hydroxylamine as energy source. Excessive growth of AOB, enhanced by applying large amounts of ammonium-fertilizer to the farmland, leads to nitrogen leaching and nitrous oxide gas emission. To suppress these unfavorable phenomena, nitrification inhibitors, AOB specific bactericides, are widely used in fertilized farmland. However, new nitrification inhibitors are desired because of toxicity and weak-effects of currently used inhibitors. Toward development of novel nitrification inhibitors that target hydroxylamine oxidoreductase (HAO), a key enzyme of nitrification in AOB, we established inhibitor evaluation systems that include simplified HAO purification procedure and high throughput HAO activity assays for the purified enzymes and for the live AOB cells. The new assay systems allowed us to observe distinct inhibitory responses of HAOs from beta-proteobacterial AOB (beta AOB) Nitrosomonas europaea (NeHAO) and gamma-proteobacterial AOB (gamma AOB) Nitrosococcus oceani (NoHAO) against phenylhydrazine, a well-known suicide inhibitor for NeHAO. Consistently, the live cells of N. europaea, Nitrosomonas sp. JPCCT2 and Nitrosospira multiformis of MOB displayed higher responses to phenylhydrazine than those of gamma AOB N. oceani. Our homology modeling studies suggest that different inhibitory responses of MOB and gamma AOB are originated from different local environments around the substrate-binding sites of HAOs in these two classes of bacteria due to substitutions of two residues. The results reported herein strongly recommend inhibitor screenings against both NeHAO of beta AOB and NoHAO of gamma AOB to develop HAO-targeting nitrification inhibitors with wide anti-AOB spectra. (C) 2016 Published by Elsevier Inc.