Purification, characterization, and catalytic mechanism of N-Isopropylammelide isopropylaminohydrolase (AtzC) involved in the degradation of s-triazine herbicides

https://doi.org/10.1016/j.envpol.2020.115803Get rights and content

Highlights

  • A native AtzC from the strain JW-1 was successfully purified and characterized.

  • Optimal conditions of the AtzC for catalyzing prometryn were 42 °C and pH 7.0.

  • AtzC, CDA, and GDA share conserved metal-binding HxH motif and His and Asp residues.

  • The structural architecture of AtzC has a single Zn2+ coordinated by His and Asp.

Abstract

Deamination is ubiquitous in nature and has important biological significance. Leucobacter triazinivorans JW-1, recently isolated from sludge, can rapidly degrade s-triazine herbicides. The responsible enzymes, however, have not been purified and characterized.

Herein, we purified an amidohydrolase, i.e., N-isopropylammelide isopropylaminohydrolase (AtzC) from JW-1 cells by ammonium sulfate precipitation and three chromatography steps. The purified AtzC catalyzed amidohydrolysis of N-isopropylammelide to cyanuric acid. The optimal catalytic conditions of the purified AtzC were 42 °C and pH 7.0, and the Km and Vmax of AtzC was 0.811 mM and 28.19 mmol/min·mg. AtzC could catalyze amidohydrolysis of an N-alkyl substituent from dihydroxy s-triazines to cyanuric acid. Molecular docking and structural alignments were used to infer AtzC catalytic mechanism. The structural architecture of AtzC resembled that of cytosine deaminase in class III amidohydrolase, with a single Zn2+ coordinated by His and Asp. Interestingly, the AtzC lacks an acidic residue putatively to activate water for hydrolysis as compared to the other amidohydrolases. His253 in AtzC probably functions as a single general acid-base catalyst. These findings further enhance our understanding how aminohydrolases catalyze the metabolism of s-triazine herbicides.

Introduction

Synthetic s-triazine herbicides (e.g., atrazine, prometryn, and terbuthylazine) have been widely used in the control of broadleaf and specific weeds all over the world (Scherr et al., 2017; Chen et al., 2018; Viegas et al., 2019). In China, the maximal residual concentration of atrazine in the soil is greater than 100 times than the allowable limit (1.0 mg/kg) established in the farmland soil after a long-term extensive application of atrazine (Liu et al., 2020). The toxicological study has shown that s-triazine herbicides can cause the disturbance of endocrine system, abnormalities of the nervous and immune system, and destruction of the reproductive system for mammals and amphibians (Hayes et al., 2011; Bohn et al., 2011;Fan and Song, 2014 ). Some of them have been banned for use in Europe, due to their contamination of the surface and groundwater (Fenoll et al., 2014). Given their high frequency of detection in soil and water (Neuwirthová et al., 2019; Quintana et al., 2019), it is a key strategy to utilize indigenous bacteria that degrade s-triazine herbicides to remove the residues. Previous studies have shown that the s-triazine herbicides can be transformed to cyanuric acid, carbon dioxide, and ammonia by degrading enzymes from bacteria (Strong et al., 2002; Mandelbaum et al., 1995; García-González et al., 2005). The most important degrading enzymes involved hydrolytic process of the s-triazine herbicides have been shown to be AtzA/TrzN, AtzB, AtzC, AtzD, AtzEG, and AtzF (Esquirol et al., 2018; Peat et al., 2017; Fan et al., 2013; Peat et al., 2015; Seffernick et al., 2010; Balotra et al., 2015a, Balotra et al., 2015b). All those enzymes belong to amidohydrolase superfamily (AHS) members. Pseudomonas sp. ADP was isolated from soil by Mandelbaum and coworkers in the 1990s (Mandelbaum et al., 1995). A series of reactions can occur under the catalysis of the degrading enzyme, including 1) hydrolytic dechlorination of the chloro-s-triazine herbicides, 2) N-dealkylation of the lateral amines, and 3) ring cleavage of cyanuric acid (García-González et al., 2005; De Souza et al., 1998). The degrading enzymes from the strain ADP can further convert chloro-s-triazine herbicides into carbon dioxide and ammonia via cyanuric acid (De Souza et al., 1996). Catabolic pathways of atrazine, the most representative triazine herbicides, are comprised of multiple hydrolases (AtzA, AtzB, AtzC, AtzD, AtzE-G, and AtzF) from Pseudomonas sp. strain ADP, which have been purified and characterized. These hydrolases are highly specific and can catalyze only s-triazine herbicides and the intermediates as substrates, which can be found in such diverse genera as Comamonas (AtzA, AtzB and AtzC) (Yang et al., 2010), Stenotrophomonas (AtzA, AtzB, AtzC and AtzD) (Galíndez-Nájera et al., 2011), Shewanella (AtzA, AtzB and AtzC) (Ye et al., 2016), Ensifer (AtzA, AtzB, AtzC, AtzD, AtzE and AtzF) (Ma et al., 2017), and Rhizobium (AtzA, AtzB and AtzC) (Fajardo et al., 2012). In addition, Arthrobacter aurescens strain TC1 and Nocardioides sp. strain C190 can use using atrazine as the sole source of nitrogen, carbon, and energy for growth in a medium containing atrazine, and can transform s-triazine herbicides into the relatively harmless cyanuric acid but not mineralize them to carbon dioxide and ammonia (Strong et al., 2002; Topp et al., 2000). The strains TC1 and C190 are lack of hydrolases such as AtzD, AtzEG, and AtzF, as a member of AHS, for further metabolizing cyanuric acid. The hydrolytic enzyme TrzN produced by A. aurescens TC1 and Nocardioides sp. C190 has similar functions to AtzA (Peat et al., 2015), which involves the first-step dechlorination of atrazine catabolism but has broader substrate specificity than AtzA (Strong et al., 2002; Topp et al., 2000). TrzN shares only a 27% sequence identity with AtzA, which is unlikely related to evolution (Meyer et al., 2009). Both TrzN and AtzA have catabolic activities to chloro-s-triazine compounds, while only TrzN has high activity to methylthio-s-triazine compounds (Shapir et al., 2005).

Generally speaking, the enzymes involved in the catabolic pathway of s-triazine herbicides are complex. Cyanuric acid is a metabolic intermediate of s-triazines herbicides, which is subsequently metabolized to ammonia and carbon dioxide by three hydrolytic enzymes (AtzD, AtzE-G or AtzE, and AtzF) (Esquirol et al., 2018; Li et al., 2004). Purification and structural characterization of AtzE produced by the model bacterium Pseudomonas sp. strain ADP have been elucidated in detail by Esquirol and colleagues (Esquirol et al., 2018), and the enzymes AtzE and AtzG have complex structures, while corrected AtzE is not a biuret amidohydrolase. Instead, the protein complex (AtzE-G) could catalyze 1-carboxybiure to remove ammonia. Additionally, some other important enzymes can convert cyanuric acid into carbon dioxide and ammonia. For example, although it is known that the specific enzyme encoded by atzH gene is composed of 129 amino acids, yet its function is still unclear. The enzyme AtzH may putatively have a transmission effect through 1-carboxybiuret to AtzE, or which can assemble into a complex with Atz E-G (Hendrickson, 2018).

Overall, most hydrolases of s-triazine herbicides in AHS have been well characterized. Notably, the catalytic mechanism of TrzN or AtzC has not been reported in detail. Seffernick et al. (2010) and Balotra et al., 2015a, Balotra et al., 2015b proposed that both TrzN and AtzC have some catalytic activities similar with the carbonic anhydrases. Due to the lack of a suitable enzyme inhibitor for crystallographic studies, the catalytic mechanism has not been well elucidated.

In our previous study, Leucobacter triazinivorans JW-1 capable of degrading prometryn, isolated from the sludge of sewage treatment plant, can effectively remove s-triazine herbicides from wastewater (Liu et al., 2017, 2018). However, how the enzymes are involved in s-triazine herbicides catabolism has not been explored. In this study, the purification and biochemical characterizations of native N-isopropylammelide isopropylaminohydrolase (AtzC) from the strain JW-1 were investigated. Meanwhile, the biochemical characteristics and substrate specificity of AtzC were studied, and the function, structure, and evolutionary relationships of AtzC were described by bioinformatics analysis. This study will be helpful for further understanding s-triazines metabolic pathways and the catalytic mechanism of AtzC.

Section snippets

Chemicals and media

Prometryn (98%), 2-hydroxypropazine (98.5%), and cyanuric acid (99%) were purchased from Dr. Ehrenstorfer GmbH (Augsburg, Germany). N-Isopropylammelide and 2,4-dihydroxy-1,3,5-triazines substrates were obtained from Hunan Chemical Research Institute (Changsha, China) and Accela ChemBio Co., Ltd (Shanghai, China), respectively. Other reagents were of analytical grade unless specified otherwise. Beef extract peptone medium (pH 7.5) consisted of 10.0 g/L peptone, 5.0 g/L beef extract, and 5.0 g/L

Purification and identification of AtzC from L. triazinivorans JW-1

The native AtzC hydrolyzing N-isopropylammelide to cyanuric acid was purified to homogeneity from cells of L. triazinivorans JW-1 using AS precipitation and three chromatography steps (HIC, SEC, and IEX). The specific enzyme activity of AtzC was determined using cyanuric acid formation and summarized in Table 1. The highest enzyme activity was found in the 60–80% of AS precipitation (Tables S–1). AtzC was purified and enriched 20.4 folds with a yield of 5.0% and a specific activity of 11.2 U/mg

Discussion

Deamination is ubiquitous in nature and has important biological significance. Among different deaminases, nonoxidative deaminases have attracted special attention on catalyzing C–N bond hydrolysis (Seffernick et al., 2001). It is well known that amidohydrolase plays a significant role in the environmental transformation of s-triazine compounds. In this study, the amidohydrolase AtzC was purified, identified, and biochemically characterized. It catalyzes the transformation of N

Conclusion

The native N-isopropylammelide isopropylaminohydrolase (AtzC) from Leucobacter triazinivorans JW-1 was successfully purified and identified by ammonium sulfate precipitation, three chromatography steps, and ESI-QTOF. The purified native AtzC catalyzes the hydrolytic transfer of an N-alkyl substituent to form cyanuric acid in the third step of prometryn catabolism in L. triazinivorans JW-1. The role of catalytic residues (His253, His60, His62, His211, Asp307) in AtzC during the process of the N

CRediT author statement

Nan Zhou: Investigation, Writing - Original draft. Jie Wang: Investigation; Data Curation. Wenbo Wang: Investigation; Visualization. Xiangwei Wu: Conceptualization; Supervision; Funding acquisition; Writing - Review & Editing.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

This work was supported in part by the National Natural Science Foundation of China (32001947 and 31572033), the Natural Science Fund for Distinguished Young Scholars of Anhui Province (1808085J16), the Natural Science Research Project of Higher Education of Anhui (KJ2017A162). We thank Dr. Qing X. Li, Rimao Hua, Pei lv, Dandan Pan and Jinjin Yao for helpful discussions and revisions.

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