An In Silico Analysis of Synthetic and Natural Compounds as Inhibitors of Nitrous Oxide Reductase (N2OR) and Nitrite Reductase (NIR)

Nitrification inhibitors are recognized as a key approach that decreases the denitrification process to inhibit the loss of nitrogen to the atmosphere in the form of N2O. Targeting denitrification microbes directly could be one of the mitigation approaches. However, minimal attempts have been devoted towards the development of denitrification inhibitors. In this study, we aimed to investigate the molecular docking behavior of the nitrous oxide reductase (N2OR) and nitrite reductase (NIR) involved in the microbial denitrification pathway. Specifically, in silico screening was performed to detect the inhibitors of nitrous oxide reductase (N2OR) and nitrite reductase (NIR) using the PatchDock tool. Additionally, a toxicity analysis based on insecticide-likeness, Bee-Tox screening, and a STITCH analysis were performed using the SwissADME, Bee-Tox, and pkCSM free online servers, respectively. Among the twenty-two compounds tested, nine ligands were predicted to comply well with the TICE rule. Furthermore, the Bee-Tox screening revealed that none of the selected 22 ligands exhibited toxicity on honey bees. The STITCH analysis showed that two ligands, namely procyanidin B2 and thiocyanate, have interactions with both the Paracoccus denitrificans and Hyphomicrobium denitrificans microbial proteins. The molecular docking results indicated that ammonia exhibited the second least atomic contact energy (ACE) of −15.83 kcal/mol with Paracoccus denitrificans nitrous oxide reductase (N2OR) and an ACE of −15.20 kcal/mol with Hyphomicrobium denitrificans nitrite reductase (NIR). The inhibition of both the target enzymes (N2OR and NIR) supports the view of a low denitrification property and suggests the potential future applications of natural/synthetic compounds as significant nitrification inhibitors.


Introduction
Nitrogen compounds are major pollutants of wastewater, owing to their involvement in the eutrophication process and their impact on the oxygen content of receiving waters. These compounds also pose toxicity risks to aquatic species (invertebrate and vertebrate) and humans [1]. Conventional nitrification/denitrification systems have been developed and applied globally to address nitrogen elimination. Ammonia elimination is generally attained by nitrifying microbes nourished on aerated surfaces in a "biological filter" [2]. Once ammonia is removed to acceptable levels by a nitrification system, an important problem is bound to arise. The combined execution of the nitrification process, along with Toxics 2023, 11, 660 2 of 16 the reduced water exchanges, causes the gradual accumulation of nitrates in recirculating aquaculture systems [3]. Nitrate (NO 3 − ) and nitrogen (N) are known to be toxic to fish at 181 mg/liter concentration levels [4]. The biological denitrification process involves the conversion of nitrate into elemental nitrogen (completing the nitrogen cycle) with the help of microorganisms. Therefore, this is an essential method required in the current scenario.
Denitrification is well understood as the dissimilatory conversion of nitrate or nitrite into a gaseous species after energy management. Nitrate accumulation is another main issue faced by intensive aquaculture practices such as recirculating aquaculture systems (RAS). The microbes involved in denitrification (denitrifiers) are aerobic, heterotrophic bacteria, with the potential to shift to an anaerobic respiration process due to anoxic conditions such as reducing NO 3 − and NO 2 − to (i) nitric oxide (NO), (ii) nitrous oxide (N 2 O), and (iii) N 2 .
The potential of denitrifiers mainly depends upon the activities of four denitrification enzymes, namely, (a) nitrate reductase (NAR); (b) nitrite reductase (NIR); (c) nitric oxide reductase (NOR); and (d) nitrous oxide reductase (N 2 OR), which are crucial in the nitrogen cycle. These enzymes are involved in the conversion of nitrous oxide (N 2 O) into N 2 . However, some denitrifiers lack these enzymes and therefore result in N 2 O as the end product. Structurally, the enzyme is a homo-dimeric protein, where the catalytic subunit of the enzyme is encoded by the nosZ gene [5]. Medicinal plants such as Mentha arvensis (essential oil), Pongamia glabra (karanja), Azadirachta indica (seed oil), and Artemisia annua have been reported to inhibit both urea hydrolysis and nitrification [6][7][8]. Zhao and colleagues [9] studied 48 plant extracts extracted with aqueous (water) and ethanol. Among these 48 plant extracts, the aqueous extracts of Epimeredi indica (aerial) and Melia azedarach (leaf) showed good urease and nitrification inhibition (NI) activities. The Pinus radiata (bark) ethanolic extract has been demonstrated to reduce nitrification, microbial biomass, carbon dioxide emissions, and urease activity [10]. Similarly, the Acacia caven (bark) and Azadirachta indica (seed kernel) extracts have been reported to inhibit urease activity [11]. Few other plants have been reported to secrete nitrification inhibitors in the rhizosphere of the soil and thus inhibit the nitrification process [12]. Similarly, legume crops, such as Arachis hypogaea (ground nut), Sorghum bicolor (sorghum), and Pennisetum glaucum (pearl millet), have been demonstrated to possess biological nitrification inhibition (BNI) in root exudate [13]. With reference, pasture grasses, such as Brachiaria decumbens and B. humidicola, have been shown to possess biological nitrification inhibition (BNI) activity via arresting both the hydroxylamine oxido-reductase [HAO] and ammonia mono-oxygenase [AMO] pathways of Nitrosomonas [13]. The phenolic root exudates of plants have been demonstrated to inhibit the nitrification process via inhibiting nitrogen-fixing bacteria, including Nitrosomonas europea [13,14]. Gallocatechin (phenolic compound) has been demonstrated to inhibit nitrification in a culture of Nitrosomonas europaea [15]. Similarly, compounds such as brachialactone [16], 1,9-decanediol [17], methyl 3-(4-hydroxyphenyl) propionate [14], safuranetin, and sorgoleone [18] have been reported to inhibit the nitrification of Nitrosomonas europaea. Caffeic acid, chlorogenic acid, condensed tannins, ellagic acid, ferulic acid, gallic acid, and hydrolysable tannins have been shown to inhibit nitrification at concentrations as low as 10 −4 to 10 −8 M [19]. Flavonoids such as isoquercitrin, myricetin, and quercetin have been shown to inhibit ammonia oxidation via ammonium-oxidizing bacteria (AOB), namely Nitrosomonas [19]. Three phenolic compounds, namely ferulic acid, vanillic acid, and tannic acid, have been demonstrated to reduce N 2 O emissions [20] via protein binding and a nitrogen immobilization mechanism [21]. Adamczyk and colleagues [22] reported that larger terpenes exhibit an identical effect of reduced soil nitrogen mineralization and nitrification, as observed with mono-terpenes. Both caffeic acid and curcumin have been reported to inhibit ammonia-oxidizing archaea (AOA), especially Nitrosomonas maritimus [23]. Allicin (from Allium species) has been reported to inhibit soil urease activity [11]. Furthermore, two sulfur compounds, namely allylsulfide and allyldisulfide (from Allium species), have been demonstrated to inhibit bacterial ammonia mono-oxygenase [AMO] activity via an irreversible inhibition mode [19]. Ferulic acid and gallic acid have been reported to inhibit biological nitrification activity by acting as outer membrane permeabilizer agents [19]. Resveratrol has been shown to inhibit the nitrification process [24]. Gao and Zhao [25] studied the efficacy of utilizing dietary phytochemicals (such as anthocyanin, gallic acid, tannin, and tannic acid) to mitigate nitrous oxide (N 2 O) emissions. Interestingly, they showed that tannin and tannic acid as dietary supplement agents reduce the nitrous oxide (N 2 O) emissions from cattle excreta by transporting their nitrogen excretion from urine to feces. However, anthocyanin and gallic acid as dietary supplement agents reduce urine nitrous oxide (N 2 O) emissions themselves. Furthermore, the mechanisms of inhibition and the potency of these compounds can differ based on the concentration, experimental conditions, and host organism [26]. In this regard, in silico screening on the mechanisms of natural and synthetic compounds is needed to fully understand the potential of these natural compounds as inhibitors of N 2 OR and NIR.

Ligand Preparation
The chemical structures of the ligands, namely The selected ligands were drawn in ChemBioDraw Ultra 12.0 and then a molecular mechanics (MM2) minimization of the ligands was performed using ChemBio3D Ultra 12.0 (www.cambridgesoft.com). Thus, these energy-minimized structures (ligands) were further utilized for the PatchDock study.

Protein Network Interaction Analysis
"The search tool for interacting chemicals" (STITCH) free-online server provides detailed information about the following; (a) metabolic pathway interactions; (b) crystal structure information; (c) binding potential; and (d) target-drug correlations [27]. In the present study, the STITCH online tool [28] was used for identifying the interactions between the twenty-two selected ligands and the proteins of the target organisms (Paracoccus denitrificans and Hyphomicrobium denitrificans).

Prediction of Insecticide-Likeness Property
In the agro-chemical discovery and development, Lipinski's rule of five (Ro5) filter was utilized to assess agro-chemical natures such as herbicides, insecticides, and pesticides. In this regard, Tice [29] adopted Lipinski's rule of five (Ro5) molecular descriptors (molecular weight; lipophilicity/hydrophobicity; number of hydrogen bond donors and acceptors; and number of rotatable bonds) as significant criteria for determining herbicidal, insecticidal, and pesticidal properties [30]. Thus, in the current study, the SwissADME free online server was used to predict the insecticide-likeness property of the selected 22 (synthetic and natural compounds) ligands [31].

Prediction of Toxicity
BeeTox is an artificial intelligence (AI)-based free online server used to predict the acute toxicity of chemicals/ligands to honey bees [32]. In the present study, the toxicity of the chosen ligands towards protozoa, bacteria (Tetrahymena pyriformis), and rodents (rat) was assessed using the BeeTox and pkCSM free online servers [31,32].

Target Protein Identification and Preparation
The three-dimensional (3D) structures of the Paracoccus denitrificans nitrous oxide reductase (PDB ID: 1FWX with a resolution of 1.6 A • ) and Hyphomicrobium denitrificans nitrite reductase (PDB ID: 2DV6 with a resolution of 2.2 A • ) were downloaded from the Research Collaborator for Structural Bioinformatics (RCSB) Protein data bank (www.rcsb. org). The "A" chains of both the selected proteins were pre-processed separately by deleting the other chains and ligands (except copper), as well as the crystallographically observed water molecules (water without hydrogen bonds). Both the proteins were prepared using the UCSF Chimera software (www.cgi.ucsf.edu/chimera) and the resultant proteins were further utilized for the PatchDock study.

PatchDock Study
The docking studies were carried out using the PatchDock free web-based server (http://bioinfo3d.cs.tau.ac.il/PatchDock). It adopts a geometry-based molecular docking algorithm method and is also utilized to recognize the binding scores, binding residues, and atomic contact energy of chosen ligands [31]. Generally, the docking results are obtained through the user's email address. We used a uniform resource locator (URL), which would provide the top 20 solutions in a table form via a user email. From these, the top one (the docked protein-ligand complex), which denoted the best solution, was selected and downloaded in the program database (PDB) file format. Finally, the binding site analyses were carried out using the PyMOL software (www.pymol.org).

Results and Discussion
Soil microbes play a vital role in nitrogen cycling, especially in terrestrial ecosystems, and they are also involved in significant transformation steps, such as nitrogen fixation, nitrification, and denitrification [26]. About 80% of the global emissions of nitrous oxide (N 2 O), which are 300 times higher than those of carbon dioxide (CO 2 ) emissions, may be due to the over-production and application of nitrogen fertilizers in the agriculture sector. Annual nitrogen fertilizer application will reach around 300 teragrams (Tg) by the year 2050, which will result in 7.5 Tg of nitrous oxide (N 2 O) emissions [33].
Nitrification inhibitors (NI) are known to alleviate the nitrate-leaching process and have also been demonstrated to decrease the nitrous oxide (N 2 O) emission rate, especially after the use of nitrogen fertilizers. In recent years, 3,4-dimethyl pyrazole phosphate (DMPP) has gained much attention among scientists and exhibited an advantage over dicyandiamide (DCD), another nitrification inhibitor [34]. Similarly, it has been reported that Fallopia species inhibit the denitrification process by releasing procyanidins via a process called BDI ("biological denitrification inhibition") [35,36].
Nitrification inhibitors using commercial compounds have minimal accessibility and an unfavorable impact on the ecosystem [37]. Herbal derivatives such as essential oils and oil cakes have been employed to block the nitrification process in soil in an ecologically safer direction [38]. A thorough literature search shows that plants and their bioactive products are capable of inhibiting the nitrification process in different soils. Sahrawat and Mukherjee [39] reported that Pongamia glabra (Indian Beech tree) seed extracts possess nitrification-inhibiting (NI) activity in different soil samples. Similarly, oil cakes derived from Citrullus colocynthis (bitter cucumber) have been reported to possess significant nitrification inhibitor activity compared to that of urea, with a 67% efficiency under both laboratory and greenhouse assays [40]. Azadirachta indica (Neem tree) seeds have been reported to exhibit the deceleration of nitrification of urea (nitrogenous fertilizer), specifically in soil with a pH of more than 6.0 [37]. Essential oils derived from Madhuca indica (Indian butter tree) and Onosma hispidum (Ratanjot) have been reported as potent nitrification inhibitors (NI) on diverse soil samples [41]. Prasad and Power [42] showed that the waste extracts of Camelia sinensis (Green tea), along with their bioactive compounds, including polyphenols, displayed a significant inhibition of soil nitrification. The flower dust derived from Chrysanthenum cinerariefolium (Pyrethrum daisy) has been reported as a potent nitrification inhibitor (NI) and to improve N use efficiency two-fold compared to prilled urea [43]. Artemisia annua (Sweet sagewort) leaf extracts containing the major metabolite artemisinin (a sesquiterpene) have exhibited significant nitrification inhibition (NI) actions on different soil samples under in vitro conditions [44]. Three native herbaceous perennial plants of Ethiopia artemis afra (Mugwort), Echinops spp. (Pale globe-thistle), and Eugenia caryophyllata (Clove) have demonstrated significant nitrification-inhibiting (NI) actions [45]. Moreover, the essential oils derived from Mentha spicata (Spearmint) have exhibited a deceleration of nitrification in the soil as compared to urea. The average NO 3 -N formation was minimal in the urea treatment compared to that of essential oils [46]. Brachiaria humidicola (Koronivia grass) root tissue extracts have been reported as nitrification inhibitors [47]. Linum usitatissimum (Linseed) essential oil has been found to exhibit nitrification-inhibiting (NI) activity [37]. Sorghum bicolor (Indian millet) root extracts showed significant nitrification inhibition (NI) under in vitro conditions in different soils [48]. Different crude extracts of Cinnamomum verum (Cinnamon), Madhuca longifolia (Madhuka), Lantana camara (Lantana), Myristica fragrans (nutmeg), and Piper nigrum (Black pepper) have shown the deceleration of nitrification with less non-target impacts on different soils [41]. Similarly, synthetic chemicals such as acetylene, azide, CO, and cyanide also act as NO inhibitors of Paracoccus denitrificans, a major nitrate-reducing microbe [49]. The above survey illustrates well that natural and chemical compounds have a greater potential to inhibit the nitrification process.
To improve the yield of crops, farmers fortify soils with different nutrients, including nitrogen fertilizers. Though, whenever applied, nitrogen fertilizers are not entirely allotted to plants. These efforts lead to the loss of diverse mechanisms such as the transformation of NO 3 − into N 2 O and N 2 through the denitrification process [50]. Earlier, Galland et al. [51] reported that the denitrification inhibition process enhances the plant growth and nutrition index of Apium graveolens L. (Celery) for a longer period. Procyanidins are polyphenolic compounds composed of condensed flavan-3-ol moieties. Procyanidins vary depending upon their monomers of (−)-epicatechin/(+)-catechin, forming oligomeric/polymeric structures, which are commonly found in apples, grapes, and sweet violets, etc. [52]. In the present study, 22 selected (synthetic and natural compounds) ligands (as shown in Table 1) were evaluated for their docking behavior with Paracoccus denitrificans nitrous oxide reductase (N 2 OR) and Hyphomicrobium denitrificans nitrite reductase (NIR) using PatchDock.                                          2-anthracene carboxylic acid azide, benzyl azide, 4-chloro-5-dimethylamino-2-phenyl-3-(2H)-pyridazinone, 3,5-dimethylpyrazole, 5-iodonaphthyl-1-azide, 1-naphthyl azide, 2-nitrophenyl azide, and phenyl azide, comply well with the TICE rule. Similarly, Table 3 shows the toxicity analysis of the selected 22 (synthetic and natural compounds) ligands, where none of the ligands have any toxicity towards honey bees. Table 3. Acute toxicity prediction of 22 selected (synthetic and natural) compounds/ligands using Bee-Tox (LabMol) and predicting small-molecule pharmacokinetic and toxicity properties (pkCSM) online tool.  Interestingly, in the present study, the STITCH analysis revealed that two ligands, namely procyanidin B2 and thiocyanate, exhibited interactions with both the Paracoccus denitrificans PD1222 ( Figure 1) and Hyphomicrobium denitrificans proteins (Figure 2), respectively. Interestingly, in the present study, the STITCH analysis revealed that two ligands, namely procyanidin B2 and thiocyanate, exhibited interactions with both the Paracoccus denitrificans PD1222 (Figure 1) and Hyphomicrobium denitrificans proteins (Figure 2), respectively.

Ligand Name
1 Figure 1. Ligand-protein interactions of (a) procyanidin B2 and (b) thiocyanate interactions with the Paracoccus denitrificans PD1222 proteins using STITCH web server.   [53]. Among these, Paracoccus denitrificans nitrous oxide reductase enzy have been exclusively reported by researchers [49]. The docking (in silico) studies binding site analyses demonstrated that arabinoxylan has the maximum atomic con energy (ACE) of −188.05 (kcal/mol), while procyanidin B1 has the lowest ACE of − (kcal/mol) with the Paracoccus denitrificans nitrous oxide reductase (as shown in Table   Table 4. Docking and interaction site analysis of 22 selected (synthetic and natural) pounds/ligands with Paracoccus denitrificans nitrous oxide reductases (N2OR) using the Patch online server.

Conclusions
In conclusion, all the selected ligands displayed docking capability with both of the targeted enzymes (N 2 OR and NIR). Interestingly among the 22 ligands, ammonia exhibited the second-lowest atomic contact energy with the nitrite reductases (NIR) of Paracoccus denitrificans and Hyphomicrobium denitrificans. The inhibition of both enzymes (N 2 OR and NIR) illustrates the nitrification inhibition potential of these 22 compounds and paves an enhanced view of the future applications of natural/synthetic compounds as significant nitrification inhibitors. Despite this, detailed in vitro screening on the mode of action of the selected compounds responsible for the denitrification process, along with microbial assays of urease activity, are required.  Data Availability Statement: All the data details are available with the corresponding authors, if any require the same can contact him for the same.

Conflicts of Interest:
The authors declare no conflict of interest.