Optimized inhibition assays reveal different inhibitory responses of hydroxylamine oxidoreductases from beta- and gamma-proteobacterial ammonium-oxidizing bacteria

https://doi.org/10.1016/j.bbrc.2016.05.041Get rights and content

Highlights

  • HAO is a key enzyme of nitrification in ammonia-oxidizing bacteria (AOB).

  • HAO is a new target of agrochemicals inhibit nitrification.

  • Assay system was optimized for HAO inhibitor development.

  • Inhibitory responses are different in two classes of βAOB and γAOB.

  • Inhibitor screenings against both βAOB and γAOB are necessary.

Abstract

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 (βAOB) Nitrosomonas europaea (NeHAO) and gamma-proteobacterial AOB (γAOB) 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 βAOB displayed higher responses to phenylhydrazine than those of γAOB N. oceani. Our homology modeling studies suggest that different inhibitory responses of βAOB and γAOB 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 βAOB and NoHAO of γAOB to develop HAO-targeting nitrification inhibitors with wide anti-AOB spectra.

Introduction

Ammonia-oxidizing bacteria (AOB), ubiquitous chemoautotrophic bacteria that convert ammonia (NH3) to nitrite (NO2) as their sole energy source, are important not only for global nitrogen cycle but also for agriculture. In farmlands, large amounts of ammonium-fertilizer are applied, and excess-grown AOB cause emission of nitrous oxide (N2O), a powerful greenhouse gas, and leaching of up to 70% of applied nitrogen from farmlands to water bodies. Therefore, nitrification inhibitors, specific bactericides for AOB, are used for increasing crop yields and/or decreasing applied nitrogen fertilizer (see review by Subbarao et al. [1]). Typical studies have shown that nitrification inhibitors decreased 63% of nitrogen leaching [2] and 38% of N2O emissions [3]. However, commercial nitrification inhibitors possess particular problems such as toxicity and weak effects. Thus, next-generation nitrification inhibitors are demanded.

Ammonia monooxygenase (AMO) and hydroxylamine oxidoreductase (HAO) are key enzymes of nitrification pathway in AOB, and catalyze ammonia (NH3) to nitrite (NO2) via hydroxylamine (NH2OH) by following chemical reactions [4]:AMO:NH3+2H++O2+2eNH2OH+H2O,HAO:NH2OH+H2ONO2+5H++4e.

In this pathway, four electrons are generated by HAO; half of the electrons are transferred back to AMO as the catalytic energy source, and the remaining half are consumed as a growth energy through generating NADPH and ATP. Therefore, both of the enzymes, AMO and HAO, are potential targets of nitrification inhibitors. Actually, AMO is believed to be a molecular target of commercial nitrification inhibitors such as nitrapyrin [5]. However, rational design of AMO-targeting inhibitor is difficult because valuable assay methods and structural information of AMO are not available yet.

HAO is a newly-emerging target of nitrification inhibitors. Recently, Wu et al. [6] have reported that the activity of soil nitrification was suppressed by organo-hydrazines that act as suicide inhibitors for HAO [7]. Crystal structures of naturally purified beta-proteobacterial AOB (βAOB) Nitrosomonas europaea HAO (NeHAO) have been solved [8], [9], [10], and they allow us to develop HAO inhibitors by structure-guided drug design. Therefore, we believe that HAO is more suitable as a nitrification-inhibitor target than AMO.

To develop nitrification inhibitors that possess wide anti-AOB spectra against a variety of uncultured soil AOB, proper model AOB strains must be selected. Okano et al. [11] showed that N. europaea is adequate to screening of nitrification inhibitors by using common nitrification inhibitors, including AMO-targeting inhibitor nitrapyrin, and βAOB strains. However, gamma-proteobacterial AOB (γAOB), which was believed to inhabit only the sea and salt lake, has been detected in soil by metagenomic analysis, recently [12]. We thus need to determine which AOB strains are adequate to screening of HAO-targeting nitrification inhibitors. In addition, a simple but effective protein purification system and high-throughput inhibitor screening/evaluation assay methods are also required to assess inhibitor response of HAO.

Here, we show different inhibitor responses of HAOs from β and γAOB by using optimized inhibitor evaluation system including simplified HAO purification methods and high-throughput inhibition assays. Our results will assist developing effective nitrification inhibitors with wide anti-AOB spectra, and will lead to promotion of sustainable agriculture.

Section snippets

Homology modeling

Homology modeling of βAOB Nitrosospira multiformis HAO (NmHAO) and γAOB Nitrosococcus oceani HAO (NoHAO) was performed using Molecular Operating Environment (MOE) software (version 2015.1001, Chemical Computing Group) with the following parameters: amino acid sequences of NmHAO (Uniprot: Q2YA36) and NoHAO (Q3JCP2), template crystal structure of NeHAO (PDB:4n4n), induced fit includes hemes and a water molecule on the iron in heme P460, forcefield of Amber10:EHT. The graphical representations

Comparison of HAOs by sequence analysis

A lot of uncultured AOB live in soil, and nitrification inhibitors should be effective to all of these AOB. Identity tree of HAO in isolated AOB (Fig. 1A) reflects classification of AOB, which are divided into two classes of βAOB and γAOB, and βAOB are further divided into two families, Nitrosomonas and Nitrosospira. We compared amino acid residues around the substrate binding pocket of HAO between βAOB and γAOB (Fig. 1B–E). Six residues arranged around the pocket on the catalytic heme P460 in

Acknowledgements

We would like to thank Dr. Wataru Tsuchiya (NARO) for technical advice, Dr. Mayumi Kuroiwa (The University of Tokyo) for protein sequence analysis. The MOE software was provided by the Agriculture, Forestry and Fisheries Research Information Technology Center (AFFRIT) of the Ministry of Agriculture, Forestry and Fisheries (MAFF), Japan. This work was in part supported by Japan Society for the Promotion of Science KAKENHI Grant 26310317 and the NIAS Strategic Research Fund to TY, and the Japan

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