Identification of the key amino acid sites of the carbofuran hydrolase CehA from a newly isolated carbofuran-degrading strain Sphingbium sp. CFD-1

Identification of the key amino acid sites of the carbofuran hydrolase CehA from a newly isolated carbofuran-degrading strain Sphingbium sp. CFD-1

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Identification of the key amino acid sites of the carbofuran hydrolase CehA from a newly isolated carbofuran-degrading strain Sphingbium sp. CFD-1 Wankui Jiang, Qinqin Gao, Lu Zhang, Hui Wang, Mingliang Zhang, Xiaoan Liu, Yidong Zhou, Zhijian Ke, Chenglong Wu, Jiguo Qiu, Qing Hong∗ Key Laboratory of Agricultural Environmental Microbiology, Ministry of Agriculture, College of Life Sciences, Nanjing Agricultural University, Nanjing, Jiangsu, 210095, PR China

A R T I C LE I N FO

A B S T R A C T

Keywords: Carbofuran Biodegradation Sphingbium sp. CFD-1 Carbofuran hydrolase CehA Key amino acid

A novel carbofuran-degrading strain CFD-1 was isolated and preliminarily identified as Sphingbium sp. This strain was able to utilize carbofuran as the sole carbon source for growth. The carbofuran hydrolase gene cehA was cloned from strain CFD-1 and expressed in Escherichia coli. CehA could hydrolyze carbamate pesticides including carbofuran and carbaryl efficiently, while it showed poor hydrolysis ability against isoprocarb, propoxur, oxamyl and aldicarb. CehA displayed maximal enzymatic activity at 40 °C and pH 7.0. The apparent Km and Kcat values of CehA for carbofuran were 133.22 ± 5.70 μM and 9.48 ± 0.89 s-1, respectively. The site-directed mutation experiment showed that His313, His315, His453 and His495 played important roles in the hydrolysis of carbofuran by CehA. Furthermore, the sequence of cehA is highly conserved among different carbofuran-degrading strains, and there are mobile elements around cehA, indicating that it may be transferred horizontally between different strains.

1. Introduction Carbofuran (2, 3-dihydro-2, 2-dimethyl-7-benzofuranyl methylcarbamate) is a broad-spectrum and highly effective carbamate insecticide, which has been widely used in the world (Chapalamadugu et al., 1992). Carbofuran is a potent inhibitor of cholinesterase in mammals (Fahmy et al., 1970; Gupta, 1994). Moreover, it also acts as an endocrine disruptor (Goad et al., 2004). Therefore, carbofuran has been banned from using in many countries, but it is still used in some developing countries due to its highly effective insecticidal action. Although carbofuran is chemically unstable due to hydrolysis in the environment, its residues are often detected in groundwater due to its widespread use in soil and high fluidity (Campbell et al., 2005), thus posing a potential hazard to non-target organisms and humans. Therefore, the environmental behavior and degradation mechanism of carbofuran have attracted increasing attention. Biodegradation, especially microbial degradation, is the main mechanism leading to the dissipation of carbofuran. To date, some carbofuran-degrading microorganisms have been reported from the genera of Achromobacterium, Flavobacterium, Pseudomonas, Sphingomonas, Novosphingobium, Paracoccus and Cupriavidus (Tomasek et al., 1989; Chaudhry et al., 1988; Rousidou et al., 2016; Yan et al., 2007, 2018;



Feng et al., 1997; Nguyen et al., 2014; Peng et al., 2008; Gupta et al., 2019). Among them, only several strains could use carbofuran as the carbon source for growth, including Sphingomonas sp. strain CF06 (Feng et al., 1997), Novosphingobium sp. KN65.2 (Nguyen et al., 2014) and Cupriavidus sp. ISTL7 (Gupta et al., 2019). So far, three genes have been cloned and functionally identified to be responsible for the hydrolysis of carbofuran, including mcd (Tomasek et al., 1989), cehA (Hashimoto et al., 2002) and mcbA (Trivedi et al., 2016; Zhu et al., 2018). Among these three genes, cehA is the earliest reported and the most widely hosted gene. In 2002, Hashimoto et al. first cloned the carbaryl hydrolase gene cehA in strain Rhizobium sp. AC100, but the catalytic ability of CehA for carbofuran was not detected (Hashimoto et al., 2002). In 2016, Rousidou et al. cloned the carbamate hydrolase gene cehA from strain Pseudmonas sp. OXA20 (Rousidou et al., 2016). Also in 2016, Öztürk et al. cloned the carbamate hydrolase gene cfdJ (99% indentity to cehA of strain AC100) from strain Novosphingbium sp. KN65.2 (Öztürk et al., 2016). In 2018, Yan et al. cloned the carbofuran hydrolase gene cehA from strain Sphingomonas sp. CDS-1 (Yan et al., 2018). Although cehA has been reported to exist in strains of different genus, its hydrolysis mechanism still needs to be investigated. In this study, strain Sphingbium sp. CFD-1, capable of degrading carbofuran and utilizing it as the sole carbon source for growth, was

Corresponding author. E-mail address: [email protected] (Q. Hong).

https://doi.org/10.1016/j.ecoenv.2019.109938 Received 31 July 2019; Received in revised form 8 November 2019; Accepted 9 November 2019 0147-6513/ © 2019 Elsevier Inc. All rights reserved.

Please cite this article as: Wankui Jiang, et al., Ecotoxicology and Environmental Safety, https://doi.org/10.1016/j.ecoenv.2019.109938

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2. Materials and methods

the cell pellet was washed three times with sterilized MSM. The optical density (OD) was adjusted to be about 2.0 at 600 nm (OD600) and used as an inoculum. Aliquots of cells (5%, v/v) were inoculated into a 250 mL Erlenmeyer flask containing 100 mL of MSM supplemented with 2.5 mM carbofuran as the sole carbon source (Sun et al., 2019). The MSM with carbofuran or strain CFD-1 only were set as two controls. All the flasks were then incubated at 30 °C with shaking at 180 rpm. The samples were collected regularly to assay the cell growth of strain CFD-1, the concentration of carbofuran and identify the metabolites. Three repetitions were used for each treatment.

2.1. Chemicals and media

2.5. Cloning of the carbofuran hydrolase gene cehA

Carbofuran (98.5%), carbaryl (98.0%), oxamyl (99.8%), aldicarb (99.7%), isoprocarb (99.2%), propoxur (98.8%) and methomyl (98.5%) were purchased from Shanghai Pesticide Research Institute. All enzymes used in DNA manipulations were obtained from Vazyme Biotech Co., Ltd. (Nanjing, China). Mineral salts medium (MSM) contained the following components (g L−1): 1.0 NaCl, 1.0 NH4NO3, 1.5 K2HPO4, 0.5 KH2PO4, 0.2 MgSO4·7H2O, pH 7.0. LB medium contained (g L−1): 10.0 tryptone, 5.0 yeast extract and 10.0 NaCl, pH 7.0. CMM was MSM supplemented with 0.2 mM carbofuran as the sole carbon source. All other chemical reagents used in this study were of the highest analytical grade.

To amplify the potential carbofuran hydrolase gene from strain CFD-1, primers (Table S2) were designed based on the nucleotide sequences of the reported carbofuran hydrolase genes including cehA, mcd and mcbA (GenBank accession numbers were NZ_NHRH01000180, AF160188 and KY123126). The flanking regions of the fragments were obtained by genomic walking strategy (Min et al., 2014). The nucleotide sequence was determined by Sangon Biotech (Shanghai) Co., Ltd. (shanghai, China). Analysis of open reading frames (ORFs) and amino acid identification were performed with ORF finder programs and BLASTX at the NCBI website (Ye et al., 2006).

isolated and its carbofuran hydrolase gene cehA was cloned and expressed. The characteristics and substrate specificity of CehA were investigated. Furthermore, four key amino acids related to carbofuran hydrolysis in CehA were also determined by site-directed mutagenesis. This is the first study to identify key amino acids in CehA from the level of pure enzyme, and investigate the detailed enzymatic properties of CehA. The results of this study help to deepen our understanding of the catalytic mechanism of CehA.

2.6. Expression and purification of recombinant CehA 2.2. Strains, plasmids, and culture conditions The expression and purification of recombinant CehA were carried out according to a method described previously (Liu et al., 2019). To express cehA in E. coli BL21 (DE3) using the pET-28a (+) system, cehA was amplified using primer pair cehA-F/cehA-R with the genomic DNA of strain CFD-1 as template (Yan et al., 2018) (Table S2). The N-terminal His-tagged CehA was purified on a Ni-NTA agarose resin matrix (Sangon Biotech, Shanghai, China). Protein concentration was determined by the Bradford method (Yang et al., 2018) with bovine serum albumin as the standard. Gel filtration chromatography was used to determine the native molecular mass of CehA. Experiments were performed at a flow rate of 0.4 mL min−1 using an AKTA purifier 10UPC system and a Superdex 200 10/300 GL column (GE Healthcare). The buffer used was 20 mM Tris-HCl buffer (pH 7.5) containing 0.1 M NaCl. The native molecular mass of CehA was estimated from a calibration curve plotted by using the standard proteins, including thyroglobulin from porcine thyroid (669 kDa), ferritin from equine spleen (440 kDa), catalase from bovine liver (232 kDa), lactate dehydrogenase from bovine liver (140 kDa), bovine serum albumin (66 kDa), and cytochrome c (12.4 kDa).

Sphingbium sp. CFD-1 was grown at 30 °C and Escherichia coli BL21 (DE3) was cultured at 37 °C. Antibiotics were used at the following concentrations: streptomycin (Str), 100 μg mL−1, ampicillin (Amp), 100 μg mL−1, kanamycin (Km), 50 μg mL−1 and gentamicin (Gm), 50 μg mL−1. Strains and plasmids used in this study are listed in Table S1. 2.3. Isolation and identification of carbofuran-degrading bacteria The activated sludge samples were collected from the wastewater treatment system of the pesticide factory in Shandong Province, P.R China. The activated sludge sample was inoculated into Erlenmeyer flask containing CMM and incubated for about 3 days at 30 °C on a 180rpm shaker. Five mL of the enriched culture was subcultured into fresh CMM every 3 days. After 4 rounds of enrichment by CMM, the carbofuran-degrading strain was isolated by spreading a serially diluted enriched culture on CMM plates. Then strain CFD-1 was obtained and used for further studies. The concentration of carbofuran was determined by UPLC and its degradation ability was confirmed. Identification of strain CFD-1 was based on Bergey's manual of determinative bacteriology (Holt et al., 1994) and sequence analysis of the 16S rRNA gene. For 16S rRNA gene sequencing and phylogenetic analysis, genomic DNA was extracted using the Bacterial Genomic DNA Mini Kit (Sangon, Shanghai, China). Universal bacterial primers 27F and 1492R (Jiang et al., 2018) were used for amplification of the 16S rRNA gene and the purified PCR product was cloned into the pMD18-T vector (Takara), then transformed into competent Escherichia coli DH5α cells and sequenced by Sangon Biotech (Shanghai) Co., Ltd. (shanghai, China). The 16S rRNA gene sequence of strain CFD-1 was compared with those available from the EzTaxon database (Kim et al., 2012). Phylogenetic analysis was performed by using the software package MEGA version 7.0 (Sudhir et al., 2017) after multiple alignments of data via CLUSTAL_X. Phylogenetic tree was reconstructed by the neighbor-joining (NJ) method (Kimura et al., 1980).

2.7. Enzyme activity assay The enzyme reaction was carried out in 1 mL of Tris-HCl buffer (20 mM, pH 7.0) at 30 °C for 10 min. The reaction mixture contained CehA (0.02 μM) and carbofuran (0.5 mM) (Hashimoto et al., 2002). One unit of enzyme activity was defined as the amount of enzyme required to hydrolyze 1 μmol carbofuran per min. Enzyme kinetic was studied using different concentrations of carbofuran (0.045 mM–1.1 mM). The substrate concentration was determined based on the integration of the chromatographic peak areas observed during the UPLC analysis. The Km and Kcat values were calculated by nonlinear regression fitting to the Michaelis-Menten equation. Each reaction was carried out in triplicate. 2.8. Biochemical properties of the recombinant CehA

2.4. Degradation of carbofuran by strain CFD-1

The optimal temperature and pH of CehA were studied using the same concentrations of CehA and carbofuran as the standard enzyme reactions. To determine the optimal reaction temperature, CehA activity was studied at temperatures between 4 °C and 60 °C. The optimal

The cells of the strain CFD-1 were cultured in liquid LB medium at 30 °C, 180 rpm for 24 h, then centrifuged at 3500×g for 5 min, and then 2

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2018). Primers cehA-F and cehA-R were used as the forward and reverse flanking primers, respectively. The internal primer pairs H351AF/R, H353A-F/R, H453A-F/R and H495A-F/R are shown in Table S2. The PCR products were gel purified, subsequently cloned into the pET28a (+) plasmid, purification of the recombinant proteins and analysis of their activity against carbofuran were performed as previously described.

pH was assayed using several buffers, including 20 mM disodium hydrogen phosphate-citric acid buffer (pH 3.0 to 7.0), 20 mM Tris-HCl (pH 7.0 to 9.0), and 20 mM glycine-NaOH buffer (pH 9.0 to 10.0). The thermostability of CehA was assessed by incubating pure enzymes at different temperatures for 2 h and then measuring the remaining activity (Zhang et al., 2017). Unheated enzyme was used as the control (100%). To determine pH stability, CehA was incubated in different pH buffers at 4 °C for 2 h and then the residual activity was measured. Samples were collected before complete consumption of carbofuran. The activity observed with the standard enzyme was defined as 100% and the relative activity of each reaction was calculated (Zhang et al., 2017). The effects of metal ions on CehA activity were determined by the addition of 1 mM different metal cations (Zn2+, Co2+, Ca2+, Li+, Fe2+, Fe3+, Hg2+, Ni2+, Mn2+, Mg2+ and Cu2+). CehA activity with no additive was referred to as the blank control and was defined as 100%, and the relative activity of CehA with different treatments was calculated. Potential enhancers and inhibitors of CehA activity were distinguished by the threshold value 100% ± 10%. The substrate specificity of CehA was determined using carbaryl, isoprocarb, propoxur, aldicarb and oxamyl. The concentration of all the substrates was 0.5 mM, and the assay was conducted in standard enzyme reaction as outlined above.

2.13. Analytical methods To analyze carbofuran and its metabolites, the reaction solution was centrifuged at 12,000×g for 5 min, and the supernatants were filtered through a 0.2-μm-pore-size filter. The carbofuran concentrations were determined using a ultra-performance liquid chromatography (UPLC) system (Dionex UltiMate 3000, USA) equipped with a C18 reverse-phase column (4.6 by 250 nm, 5 μm). The mobile phase was composed of methanol-water (75:25, v/v) at a flow rate of 0.8 mL min−1. The detection wavelength was 280 nm, column temperature was maintained at 40 °C and the sample volume was 20 μL. For identification of the metabolites, the mass spectrum was collected using a TripleTOF 5600 (AB SCIEX) mass spectrometer. The metabolites were ionized by electrospray with positive polarity, and characteristic fragment ions were detected using MS/MS. The mass spectra of the authentic carbofuran and carbofuran phenol were analyzed under the same conditions, and the results were used as the standards. As the authentic 4-hydroxy carbofuran phenol (compound III) and 2, 2-dimethyl-2, 3-dihydrobenzofuran-4, 7-dione was not available (compound IV), the analysis of its MS/MS fragments was also shown in Fig. S1. To investigate the substrate specificity of CehA, the activity of CehA against carbaryl, propoxur and isoprocarb, their concentrations were determined using the same UPLC system, and the detection condition was the same as that of carbofuran. Although oxamyl, aldicarb and methomyl were also determined by the same UPLC system, the mobile phase was different and it consisted of acetonitrile-water (50:50, v/v) at a flow rate of 0.8 mL min−1. The column temperature was set at 40 °C. Column elution was monitored by measuring the absorbance at 243 nm.

2.9. Construction of cehA gene-disrupted strain CFD-1 To disrupt cehA through a single-crossover procedure (Gu et al., 2013), a 600-bp DNA fragment (in the middle of cehA) was amplified from strain CFD-1 with primers KcehA-F and KcehA-R (Table S2). The fragment was cloned into the EcoRI-and BamHI-digested pEX18Gm plasmid (Hoang et al., 1998) using the ClonExpressII one-step cloning kit to produce pEX-cehA. The corresponding plasmid was introduced into the strain CFD-1 by electroporation, as described by Zhang et al. (2011). Single-crossover clones were selected on LB plates supplemented with streptomycin, 100 μg mL−1, and gentamicin, 50 μg mL−1. The cehA insertion mutant, designated CFD-KA, was verified by PCR with primers KV-F and KV-R (Table S2), and the resulting amplicons were sequenced to confirm the successful integration of pEX-cehA in strain CFD-KA. Its ability to degrade carbofuran was tested in MSM supplemented with 0.5 mM carbofuran.

2.14. Accession number(s)

2.10. Complementation of the cehA-disrupted mutant

The 16S rRNA gene sequence and the DNA fragment (6506-bp) containing the carbofuran hydrolase gene cehA from strain CFD-1 were deposited in the GenBank database under accession numbers of MK817560 and MK988315, respectively.

A 2298-bp fragment containing the 297-bp region upstream of cehA was amplified from strain CFD-1 using primers MCS-CF and MCS-CR (Table S2). The PCR product was cloned into the EcoRI- and BamHI -digested broad-host-range plasmid pBBR1MCS-2 (Kovach et al., 1995) using the ClonExpressII one-step cloning kit to produce pBBR1-cehA, which was then introduced into the mutant strain CFD-KA through electroporation to generate CFD-KA(pBBR1-cehA). The strain's ability to degrade carbofuran was assayed in MSM supplemented with 0.5 mM carbofuran.

3. Results and discussion 3.1. Isolation and characterization of the carbofuran-degrading strain A bacterial strain CFD-1 capable of degrading carbofuran was isolated using the enrichment method. It was Gram-negative and rodshaped (0.6–0.8 μm × 1.6–2.2 μm) and non-flagellate. Colonies of strain CFD-1 were yellowish, circular, convex and smooth when growing at 30 °C on LB plate (data not shown). Strain CFD-1 was positive for the following activities or reactions: Ala-Phe-Pro arylamidase, glutamyl arylamidase pNA, L-proline arylamidase, tyrosine arylamidase. It was negative for oxidase, urease, nitrate reduction, and utilization of D-glucose, L-arabinose, D-mannose, maltose, citrate, mannitol, gluconate, phenylacetic acid, adipate and N-acetylglucosamine. The 16S rRNA gene sequence of strain CFD-1 was a continuous stretch of 1451 bp (MK817560), and it was highly similar to those of known Sphingobium strains, including Sphingobium baderi LL03T (98.9%) (Kaur et al., 2013), Sphingobium faniae JZ-2T (98.5%) (Guo et al., 2010), Sphingobium wenxiniae JZ-1T (98.5%) (Wang et al., 2011). The phylogenetic tree showed that strain CFD-1 was located in the same cluster with Sphingobium baderi LL03T (JN695620), Sphingobium faniae

2.11. DEPC inhibition DEPC is a histidine inhibitor, which is able to inhibit some enzymes with a histidine at the active center. To determine the role of histidine in CehA, it was subjected to the treatment of DEPC. CehA was treated with 1 mM DEPC at 4 °C for 2 h and then dialyzed against 20 mM TrisHCl (pH 7.5) to remove the DEPC (Wu et al., 2017). Enzyme activity was determined as described above and compared to the activity of CehA without DEPC treatment. 2.12. Histidine mutation Point mutations in CehA gene were constructed by overlap PCR according to the standard site-directed mutagenesis protocol (Zhu et al., 3

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Sphingmononas sp. CDS-1 (Yan et al., 2018). The initial step is the hydrolysis of carbofuran to carbofuran phenol and methylcarbamic acid, then the latter spontaneously hydrolyzed to methylamine and CO2. However, neither methylcarbamic acid nor methylamine was directly detected by UPLC, possibly because they cannot absorb UV light. Carbofuran phenol can be further transformed to 4-hydroxy carbofuran phenol by strain CFD-1. Next, it might be further catalyzed by a series of enzymes, and the yielding metabolite finally entered into the TCA cycle, although the related products were not detected through the UPLC, the growth of strain CFD-1 with carbofuran phenol as the carbon source supports this speculation (data not shown). 3.3. Cloning and sequence analysis of the cehA gene Three pairs of primers were used for the amplification of the carbofuran hydrolase genes (cehA, mcd and mcbA). Only the cehA gene (2385-bp) was successfully amplified from strain CFD-1 (Fig. S4). Subsequently, a 6506-bp DNA fragment flanking this 2385-bp region was obtained by genome walking. The physical map of this fragment was shown in Fig. 3, and analysis of its sequence by the online ORF Finder and BLASTx (www.ncbi.nlm.nih.gov) identified three complete ORFs, designated as TonB-dependent receptors, cehA and IS6100. The protein sequences encoded by these three ORFs were used as queries in the BLASTP search (UniProtKB/Swiss-Port database) and functions were proposed for each ORF (Table S3). CehA showed 99% identity to the carbofuran hydrolase (CehA) from Sphingomonas sp. CDS-1 (WP087575984) (Yan et al., 2018), 99% identity to the carbamate hydrolase (CfdJ) from Novosphingobium sp. KN65.2 (BAB85626) (Nguyen et al., 2014), 99% identity to the carbaryl hydrolase (CehA) from Rhizobium sp. AC100 (CDO34164) (Hashimoto et al., 2002), 99% identity to the putative carbamate hydrolase (CehA) from Pseudomonas sp. OXA20 (CBY85381) (Rousidou et al., 2016). In strain CFD-1, a putative TonB-dependent receptor is located in the upstream of cehA, while an IS element (IS6100) is located in its downstream. As the first discovered and currently the most widely hosted carbamate hydrolase gene, cehA is widely found in the natural environment contaminated by carbamate pesticide (Rousidou et al., 2017), and its sequence is highly conserved among different carbofuran-degrading strains. Also there are mobile elements around cehA, indicating that it may be transferred horizontally between different strains.

Fig. 1. Utilization of carbofuran as the sole carbon source for growth by Sphingobium sp. CFD-1. ○, carbofuran abiotic control; , cell density of strain CFD-1 without carbofuran; ●, carbofuran with strain CFD-1; , cell density of strain CFD-1 with carbofuran. Cell growth was determined by the colony counting method. Error bars represent the standard errors from three replicates.

JZ-2T (FJ373058), Sphingobium wenxiniae JZ-1T (FJ686047) (Fig. S2). Based on the morphological, physiological characteristics and the analysis of 16S rRNA gene sequences, strain CFD-1 was preliminarily identified as Sphingobium sp., which is a new carbofuran-degrading strain isolated from the genus of Sphingobium. 3.2. Degradation of carbofuran by strain CFD-1 The degrading of carbofuran and growth of strain CFD-1 were simultaneously investigated in MSM with 2.5 mM of carbofuran as the sole carbon source (Fig. 1). The growth of strain was accompanied by the degradation of carbofuran. About 95% of carbofuran was degraded after incubation for 60 h, and the cell density had increased from 0.18 × 108 CFU mL−1 to 1.20 × 108 CFU mL−1. However, a slight decrease in the cell density of CFD-1 (from 0.18 × 108 CFU mL−1 to 0.175 × 108 CFU mL−1) was observed in the medium without carbofuran as the carbon source, and no significant change in carbofuran concentration was observed in the uninoculated culture. Also, after the degradation of carbofuran by strain CFD-1, the cells were collected, and then subjected to the extraction of carbofuran by sonication and the detection of carbofuran, the results showed that no carbofuran was detected, indicating that no carbofuran was adsorbed by the cells of strain CFD-1. All of these revealed that CFD-1 can degrade carbofuran and utilize it as the sole carbon source for growth. Samples for metabolite detection were collected at different time point after inoculation. Four compounds (compounds I, II, III and IV) were detected by UPLC (Fig. S3) with retention times of 5.11 min, 5.74 min, 3.87 min and 4.27 min, respectively. The sample collected at 24 h was selected for mass analysis because it contained the proper quantity of all four compounds (Fig. 2A). The main protonated molecular ion m/z of compound I was 222.1123 [M+H]+, which was identified as carbofuran (C12H16NO3+, m/z 222.1125) with an error of 0.9 ppm (Fig. 2B). The main protonated molecular ion m/z of compound II was 165.0911 [M+H]+, which was consistent with carbofuran phenol (C10H13O2+, m/z 165.0910), with a 0.6-ppm error (Fig. 2C). The molecular ion m/z of compound III was 181.0856 [M+H]+, which corresponds to 4-hydroxy carbofuran phenol (C10H13O3+, m/z 181.0859), with a 1.7-ppm error (Fig. 2D). The molecular ion m/z of compound IV was 179.0698 [M+H]+, which corresponds to 2, 2-dimethyl-2, 3-dihydrobenzofuran-4, 7-dione (C10H13O3+, m/z 179.0703), with a 2.8-ppm error (Fig. 2E). Generally, a mass error between −5 ppm and 5 ppm is acceptable for the identification of compounds (Blake et al., 2011). Therefore, a putative degradation pathway of carbofuran in strain CFD-1 was proposed based on the results of this study (Fig. 2F). This pathway is the same as that of

3.4. Heterologous expression of cehA In order to further study the catalytic activity of cehA, the pure enzyme CehA was obtained by heterologous expression. Purified CehA was observed as a single band on SDS-PAGE, its approximate size was in agreement with its theoretical mass (87.66 kDa) derived from its amino acid sequence (Fig. S5). Thus, we deduced that CehA exists naturally as a homodimer (Fig. S6). CehA catalyzed the cleavage of ester bond in carbofuran to produce carbofuran phenol and methylcarbamic acid, which then spontaneously hydrolyzed to methylamine and CO2 (Rousidou et al., 2016; Yan et al., 2018). The enzyme assay of CehA showed that it hydrolyzed carbofuran to produce carbofuran phenol (Fig. 4). This is the only product detected in this assay. Therefore, CehA is responsible for the hydrolysis of carbofuran, which is the initial step in the degradation of carbofuran in strain CFD-1. 3.5. Biochemical characterization of CehA The specific activity of CehA is 5.22 U mg−1 for carbofuran, while the Vmax, Km, and Kcat of CehA for carbofuran are 0.087 ± 0.017 μmol s-1 mg-1, 133.22 ± 5.70 μM and 9.48 ± 0.89 s-1, respectively (Fig. S7). The optimum temperature and pH for catalysis is 40 °C and 7.0, respectively (Fig. S8). However, since the stability of enzyme for CehA was low at 40 °C, subsequent enzymatic experiments were performed at 30 °C. CehA activity was enhanced by Cu2+, Co2+, Zn2+ and Ni2+, 4

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Fig. 2. Degradation pathway of carbofuran in strain CFD-1. (A) UPLC analysis of metabolites that appeared during the degradation of carbofuran by strain CFD-1. Samples for metabolite detection were collected 24 h after inoculation. The conditions for degradation were 30 °C, pH 7.0, in MSM with 2.5 mM of carbofuran as the sole carbon source and the initial inoculum was 0.18 × 108 CFU mL−1 of cells. Four compounds (I, II, III and IV) were detected with retention times of 5.11 min, 5.74 min, 3.87 min and 4.27 min. (B) MS analysis of compound I (m/z 222.1123 [M+H]+), which was identified as carbofuran. (C) MS analysis of compound II (m/z 165.0911 [M+H]+), which was identified as carbofuran phenol. (D) MS analysis of compound III (m/z 181.0856 [M+H]+), which was identified as 4-hydroxy carbofuran phenol. (E) MS/MS analysis of compound IV (m/z 179.0698 [M+H]+), which was identified as 2, 2-dimethyl-2, 3-dihydrobenzofuran-4, 7-dione. (F) The proposed metabolic pathway of carbofuran in strain CFD-1.

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methomyl, its side chain is relatively simple than those of other carbamate insecticides in the present study, and cehA showed not detectable activity against it. However, the specific mechanism needs further study in the future. In comparison with other reported carbamate hydrolases (McbA, CehA and CfdJ), the substrate specificity of CehA in strain CFD-1 was similar to that of CehA in strain AC100, CfdJ in strain KN65.2 and McbA in strain XWY-1 (Hashimoto et al., 2002; Zhu et al., 2018; Öztürk et al., 2016), but was broader than that of McbA, as McbA showed no activity against oxamy (Zhu et al., 2018). Therefore, CehA has good potential for the hydrolysis of carbamate pesticides.

Fig. 3. Genomic context of cehA in strain CFD-1. The arrows indicate the size, location, and transcription direction of the ORFs.

3.7. The cehA gene is essential for the degradation of carbofuarn To verify whether cehA is the only gene involved in the initial degradation step of carbofuran by strain CFD-1, cehA was disrupted through a single-crossover event. The resulting mutant strain, CFD-KA, lost the ability to hydrolyze carbofuran. The complemented strain CFDKA (pBBR1-cehA) regained the ability to hydrolyze carbofuarn (Fig. S9). Strain CFD-KA (pBBR1-cehA) not only regained the ability to hydrolyze carbofuran to carbofuran phenol but also further degraded carbofuran phenol, the metabolites detected by UPLC were the same as those appeared during the degradation of carbofuran by the wild strain CFD-1. Taken together, these results showed that cehA was essential for the degradation of carbofuran by strain CFD-1.

Fig. 4. UPLC analysis of hydrolysis product of carbofuran by CehA. The enzyme reaction was carried out in 1 mL of Tris-HCl buffer (20 mM, pH 7.0) containing CehA (0.02 μM) and carbofuran (0.5 mM) at 30 °C for 10 min. A and C indicate the standard samples of carbofuran (RT = 5.11 min) and carbofuran phenol (RT = 5.74 min), respectively. B indicates the hydrolysis of carbofuran by CehA.

3.8. Identification of key amino acids sites for catalytic activity of CehA The treatment of DEPC inhibited the activity of CehA by 86% (data not shown), and the result showed that DEPC had significant inhibitory effect on the activity of CehA, indicating that histidine might be involved in the catalytic activity of CehA. To further determine which histidine plays a crucial role in CehA, its amino acid sequences were compared with those of another carbaryl hydrolase McbA, which is also an esterase (Peng et al., 2008), and it was speculated that these four histidine residues (His351, His353, His453 and His459) might be catalysis-related key amino acid sites in CehA. In order to verify this speculation, four mutants were obtained by replacing the histidine residues with alanine residues, yielding CehAH351A (His351 to Ala351), CehAH353A (His353 to Ala353), CehAH453A (His453 to Ala453) and CehAH495A (His495 to Ala495) (Fig. S5). The enzyme activity assay showed that all of the variants lost hydrolysis activity against carbofuran, indicating that these four histidine residues were key amino acid sites of the carbofuran hydrolase CehA. The amino acid sequence alignment analysis of CehA with its closely related esterases revealed that the catalytic triad SDH/SEH (Reva et al., 2002) of esterase was also

while it was inhibited by Fe3+ and Hg2+. Additionally, Fe2+, Ca2+, Mn2+and Mg2+had no obvious effects on CehA activity (Table S4).

3.6. Substrate specificity of CehA In addition to carbofuran, CehA was also able to hydrolyze different carbamate pesticides including carbaryl, isoprocarb, propoxur, aldicarb and oxamyl, with different levels of efficiency. Carbaryl was hydrolyzed faster than carbofuran, while the relative activity against isoprocarb, propoxur, aldicarb and oxamyl was lower than that for carbofuran. And no activity was detected against methomyl (Table S5). Carbamate insecticides are a class of insecticides containing a common carbamate structure and a different side chain, and CehA showed different catalytic effect on different substrates (Table S5). We speculate that the catalytic pocket of cehA might prefer to bind to carbamate insecticides with more complex side chain structure. As for

Fig. 5. Alignment of amino acid sequences of CehA from strain CFD-1 with the most closely related proteins available in the UniProtKB/Swiss-Prot database (amino acid sequences from 298 to 526 of CehA). The related functional protein: MK988315, CehA from Sphingbium sp. CFD-1; BAB85626, CehA from Rhizobium sp. AC100; WP087575984, CehA from Sphingomonas sp. CDS-1; CDO34164, CfdJ from Novsphingbium sp. KN65.2; CBY85381, CehA from Pseudmonas sp. OXA20; AJR27119, DdhA from Sphingobium sp. YBL2; WP104963089, McbA from Pseudmonas sp. XWY-1. The conserved sequence signature (G-X-S-X-G) is marked with the blue background; the catalytic triad S-D-H (Ser347, Asp349, His351) are indicated with the green background. * indicates that these sites are amino acids involved in the mutations in this study. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.) 6

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present in CehA (Fig. 5 shows the alignment results of amino acid sequences from 298 to 526, and Fig. S10 shows alignment results of complete amino acid sequences), while the conserved and consensus sequence of esterase G-X1-S-X2-G (Ollis et al., 1992) is only present in McbA and DdhA (Zhu et al., 2018; Yan et al., 2016) but not in CehA, and the phylogenetic analysis also showed that CehA formed an independent branch on the phylogenetic tree (Fig. S11). All of these indicated the sequence uniqueness of CehA. Among the four mutated histidine residues (His351, His353, His453 and His459), H351 is in the catalytic triad of SDH, H453 is in the region G-X1-L-X2-L of CehA, which is different from the typical conserved region G-X1-S-X2-G of esterase, but H453 is in the typical G-X1-S-X2-G region at the corresponding positions of McbA and DdhA (Fig. 5), suggesting that the characteristic sequence of esterase may has changed in CehA, because CehA has a low amino acid sequence similarity to the other esterases. As for the variants derived from the mutation at H353 and H459, they also lost the enzyme activity. However, the reason for this needs to be further studied in the future in combination with the crystal structure analysis of CehA.

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4. Conclusion A new carbofuran-degrading strain Sphingbium sp. CFD-1 was isolated, which could utilize carbofuran as a sole carbon source for growth. The metabolic pathway of carbofuran in strain CFD-1 was analyzed. The carbofuran hydrolase gene cehA was cloned and expressed in E. coli. The optimal pH and temperature for CehA to hydrolyze carbofuran was 7.0 and 40 °C, respectively. CehA had a broad substrate spectrum for carbamate pesticides. Four histidine residues (His351, His353, His453 and His495) were identified as the crucial amino acid sites of CehA. This study enriches the diversity of carbofuran-degrading bacteria and provides an important theoretical basis for the enzymatic mechanism of carbamate hydrolases. Declaration of competing interest The authors declare that they have no competing interests. Acknowledgements This work was supported by the National Natural Science Foundation of China (31670112, 31970102), the National Key R & D Program of China (2017YFD0800702) and the Postgraduate Research & Practice Innovation Program of Jiangsu Province (KYCX18_0744). Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.ecoenv.2019.109938. References Blake, S.L., Walker, S.H., Muddiman, D.C., Hinks, D., Beck, K.R., 2011. Spectral accuracy and sulfur counting capabilities of the LTQ-FT-ICR and the LTQ-Orbitrap XL for small molecule analysis. J. Am. Soc. Mass Spectrom. 22, 2269–2275. Campbell, S., David, M.D., Woodward, L.A., Li, Q.H., 2005. Persistence of carbofuran in marine sand and water. Chemosphere 54, 1155–1161. Chapalamadugu, S., Chaudhry, G.R., 1992. Microbiological and biotechnological aspects of metabolism of carbamates and organophosphates. Crit. Rev. Biotechnol. 12, 357–389. Chaudhry, G.R., Ali, A.N., 1988. Bacterial metabolism of carbofuran. Appl. Environ. Microbiol. 54, 1414–1419. Fahmy, M.A., Fukuto, T.R., Myers, R.O., March, R.B., 1970. The selective toxicity of new N-phosphorothioyl-carbamate esters. J. Agric. Food Chem. 18, 793–796. Feng, X., Ou, L.T., Ogram, A., 1997. Plasmid-mediated mineralization of carbofuran by Sphingomonas sp. strain CF06. Appl. Environ. Microbiol. 63, 1332–1337. Goad, R.T., Goad, J.T., Atieh, B.H., Gupta, R.C., 2004. Carbofuran-induced endocrine disruption in adult male rats. Toxicol. Mech. Methods 14, 233–239. Gu, T., Zhou, C., Sørensen, S.R., Zhang, J., He, J., Yu, P., Yan, X., Li, S., 2013. The novel bacterial N-demethylase PdmAB is responsible for the initial step of N, N-dimethyl-

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