Carbamate C-N Hydrolase Gene ameH Responsible for the Detoxification Step of Methomyl Degradation in Aminobacter aminovorans Strain MDW-2

Based on the structural characteristic, carbamate insecticides can be classified into oxime carbamates (methomyl, aldicarb, oxamyl, etc.) and N-methyl carbamates (carbaryl, carbofuran, isoprocarb, etc.). So far, research on the degradation of carbamate pesticides has mainly focused on the detoxification step and hydrolysis of their carbamate bond. Several genes, such as cehA, mcbA, cahA, and mcd, and their encoding enzymes have also been reported to be involved in the detoxification step. However, none of these enzymes can hydrolyze methomyl. In this study, a carbamate C-N hydrolase gene, ameH, responsible for the detoxification step of methomyl in strain MDW-2 was cloned and the key amino acid sites of AmeH were investigated. These findings provide insight into the microbial degradation mechanism of methomyl. ABSTRACT Methomyl {bis[1-methylthioacetaldehyde-O-(N-methylcarbamoyl)oximino]sulfide} is a highly toxic oxime carbamate insecticide. Several methomyl-degrading microorganisms have been reported so far, but the role of specific enzymes and genes in this process is still unexplored. In this study, a protein annotated as a carbamate C-N hydrolase was identified in the methomyl-degrading strain Aminobacter aminovorans MDW-2, and the encoding gene was termed ameH. A comparative analysis between the mass fingerprints of AmeH and deduced proteins of the strain MDW-2 genome revealed AmeH to be a key enzyme of the detoxification step of methomyl degradation. The results also demonstrated that AmeH was a functional homodimer with a subunit molecular mass of approximately 34 kDa and shared the highest identity (27%) with the putative formamidase from Schizosaccharomyces pombe ATCC 24843. AmeH displayed maximal enzymatic activity at 50°C and pH 8.5. Km and kcat of AmeH for methomyl were 87.5 μM and 345.2 s−1, respectively, and catalytic efficiency (kcat/Km) was 3.9 μM−1 s−1. Phylogenetic analysis revealed AmeH to be a member of the FmdA_AmdA superfamily. Additionally, five key amino acid residues (162, 164, 191, 193, and 207) of AmeH were identified by amino acid variations. IMPORTANCE Based on the structural characteristic, carbamate insecticides can be classified into oxime carbamates (methomyl, aldicarb, oxamyl, etc.) and N-methyl carbamates (carbaryl, carbofuran, isoprocarb, etc.). So far, research on the degradation of carbamate pesticides has mainly focused on the detoxification step and hydrolysis of their carbamate bond. Several genes, such as cehA, mcbA, cahA, and mcd, and their encoding enzymes have also been reported to be involved in the detoxification step. However, none of these enzymes can hydrolyze methomyl. In this study, a carbamate C-N hydrolase gene, ameH, responsible for the detoxification step of methomyl in strain MDW-2 was cloned and the key amino acid sites of AmeH were investigated. These findings provide insight into the microbial degradation mechanism of methomyl.

water due to its high water solubility (58 g liter Ϫ1 ) and low affinity for sorption to soils (5,6). Recently, methomyl has been identified to be a strong genotoxic agent and an agent inducing cell DNA damage and apoptosis in vitro (7). The WHO (World Health Organization), EPA (Environmental Protection Agency), and EC (European Commission) have also declared methomyl to be a toxic and hazardous pesticide (8). Although the use of methomyl has been banned in Europe and the United States for several years, it is still used in some developing countries (9). Therefore, the environmental behavior and degradation mechanisms of methomyl are of great concern and interest.
Aminobacter aminovorans MDW-2, a methomyl-degrading strain utilizing methomyl as the sole carbon source for growth, was previously isolated (27). In the present study, we focused on cloning the carbamate C-N hydrolase gene ameH, responsible for the hydrolysis of the amide bond of methomyl in strain MDW-2, which is a common detoxification degradation step in all microorganisms. Additionally, the characteristics and key amino acid sites of AmeH were investigated. The study results suggest that ameH is essential for the degradation of methomyl, which will provide insight into the methomyl degradation mechanism.
four purification steps, the specific activity of AmeH increased to 222.2 U/mg and the protein was concentrated 11.8-fold with 12.5% recovery (Table S1). The molecular mass of the protein on ExpressPlus PAGE gels was about 30 kDa (Fig. S2). Preliminary identification of methomyl degradation was carried out by high-performance liquid chromatography (HPLC). The protein band was then excised and analyzed by matrixassisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectrometry, and the amino acid sequences of the peptide fragments were obtained.
The whole genome of strain MDW-2 was sequenced to identify the gene encoding AmeH. The analysis revealed four replicons, consisting of one circular chromosome (5, Fig. S3). The whole genome had an average GϩC content of 63.2%. A total of 6,799 protein-coding genes were predicted. The amino acid sequences of the abovementioned peptide fragments were then compared with all annotated proteins from the strain MDW-2 genome. The sequences were matched with a hydrolase encoded on orf1623 (951 bp) (Fig. S4) in pMDW1; this gene was designated ameH and selected for further study. The digital DNA-DNA hybridization (dDDH) between the genomes of strain MDW-2 (CP060197 to CP060200) and Aminobacter aminovorans DSM 7048 T (ASM434164v1) was 78.3%, higher than the standard species boundary for dDDH (70%). The results indicated that strain MDW-2 could be identified as the same species as Aminobacter aminovorans DSM 7048 T . Therefore, strain MDW-2 was identified as Aminobacter aminovorans MDW-2.  (30). The phylogenetic analysis based on the amino acid sequences of AmeH and closely related proteins revealed that AmeH is a member of the FmdA_AmdA superfamily and could form a clade with other amidases within the FmdA_AmdA superfamily, suggesting a close evolutionary relationship with amidases within the FmdA_AmdA superfamily (Fig. 2). These results indicate that AmeH constitutes a novel carbamate C-N hydrolase within the Fm-dA_AmdA superfamily.
Heterogeneous expression of ameH. The ameH gene was cloned and expressed in Escherichia coli BL21(DE3) to further investigate the catalytic activity of AmeH on the hydrolysis of methomyl. The yield of purified AmeH-6His was approximately 3.8 Ϯ 0.2 mg per liter of culture. The molecular mass of native AmeH-6His was calculated to be 65 kDa by gel filtration chromatography. The purified AmeH-6His appeared as a single band on SDS-PAGE with a molecular mass of 34 kDa. Therefore, we inferred that AmeH-6His exists as a homodimer naturally (Fig. S5).
HPLC-tandem mass spectrometry (MS/MS) was employed to identify the hydrolysis product of methomyl by AmeH-6His in the enzyme assay. Two compounds were detected with retention times of 3.63 min (compound I) and 4.01 min (compound II) (Fig. 3A). The prominent protonated molecular ion of compound I was m/z 163.0534 [MϩH] ϩ , which was identified as methomyl (C 5 H 11 N 2 O 2 S ϩ , m/z 163.0536) with a 1.2-ppm error (Fig. 3B). The molecular ion mass of compound II was m/z 106.0322 [MϩH] ϩ , which was consistent with the protonated derivative of methomyl oxime (C 3 H 8 NOS 2 ϩ , m/z 106.0321) with a 0.6-ppm error (Fig. 3C). Generally, a mass error between Ϫ5 ppm and 5 ppm is acceptable for the identification of compounds (31). These results indicated that AmeH-6His hydrolyzed the amide bond of methomyl to produce methomyl oxime (Fig. 3D), which was different from the catabolism pathway reported previously in the degradation of methomyl by strain MDW-2 (27) (Fig. S1). The reason is addressed in Discussion.

FIG 2
Phylogenetic tree constructed based on the alignment of AmeH with related proteins. The multiple-alignment analysis was performed with ClustalX 2.1 software. The neighbor-joining method was used to construct the unrooted phylogenetic tree through MEGA 7.0. Bootstrap percentages (based on 1,000 replications) of Ͼ50% are shown on the branches. According to their respective amino acid sequence and function, the clustering of amidases is displayed in different colors. The green, blue, and purple colors correspond to the FmdA_AmdA superfamily, nitrilase superfamily, and amidase signature family, respectively. ForM, formamidase; AceM, acetamidase; Ami, amidase; ALAM, aliphatic amidase. In the FmdA_AmdA superfamily clan, accession numbers are as follows: AAN87355, however, at a higher temperature, 70°C, the residual AmeH-6His activity fell below 10% in 1 h (Fig. S7B). AmeH-6His exhibited high activity levels at a pH range of 6.5 to 9.0, and retained 75% of its activity after storage at pH 7.0 to 8.5 for 1 h at 30°C (Fig. S8B).
ameH is essential for degradation of methomyl. The ameH gene was disrupted by a single-crossover event to verify whether it is the only gene involved in the detoxification degradation step of methomyl in strain MDW-2. The resulting mutant, strain MDW-2M, lost the ability to hydrolyze methomyl, while the complemented strain MDW-2M (pBBR1-ameH) regained the ability to degrade methomyl (Fig. S9). These results confirmed that AmeH is responsible for hydrolysis of the amide bond of methomyl, which is the detoxification step of methomyl degradation in strain MDW-2.
Identification of key amino acid sites of AmeH. Alignment of the amino acid sequences of AmeH and closely related proteins (identity, 26% to 33%) whose crystal structures are available in the protein data bank (PDB) revealed 26 conserved amino acid sites (indicated by shading in Fig. S10). Of these, five were predicted to be key amino acids in 3TKK_A (an acetamidase from Thermotoga maritima), corresponding to the residues G162, N164, D191, H193, and E207 in AmeH (indicated by green diamonds in Fig. S10) (32). To verify this further, the five amino acids were individually replaced by alanine in AmeH, and five variants (AmeH-6His G162A, AmeH-His N164A, AmeH-His D191A, AmeH-His H193A, and AmeH-His E207A) were obtained (Fig. S11). The yield of purified variants was estimated to be approximately 3.8 mg per liter of culture. The enzyme activity assay showed that only AmeH-His H193A retained 10% of AmeH-6His hydrolysis activity against methomyl, while the activity in the other four variants was abolished entirely (Table. S4). The results confirmed these five amino acids to be the key residues of AmeH-6His.

DISCUSSION
To date, many bacterial strains capable of degrading carbamate insecticides have been reported from the genera Stenotrophomonas, Achromobacter, Flavobacterium, Pseudomonas, Sphingomonas, Novosphingobium, Paracoccus, Aminobacter, and Cupriavidus (11)(12)(13)(14)(15)(16)(17)(18)(19)23). The molecular basis of degradation of certain carbamate insecticides has been studied extensively. Four carbamate hydrolase genes, cehA, mcd, cahA, and mcbA, have been cloned so far (11,13,14,22,33). Among the reported carbamate hydrolases, only McbA has been proven to have no catalytic activity against methomyl (22). However, the activities of the other three enzymes (CehA, Mcd, and CahA) against methomyl have not been studied so far. Therefore, in this study, the heterologous expression of CehA, Mcd, and CahA was carried out, and their activity on methomyl was determined. The results demonstrated that these enzymes could not hydrolyze methomyl (the experimental methods and related conclusions are shown in Text S1 in the supplemental material). None of the known carbamate hydrolases could hydrolyze methomyl. In this study, ameH was cloned from the methomyl-degrading strain Aminobacter aminovorans MDW-2. Although AmeH has relatively good hydrolysis activity for methomyl, it showed a narrow substrate spectrum and no activity against other carbamate insecticides (Table S3).
In this study, AmeH hydrolyzed methomyl to methylamine and an unstable compound that spontaneously transformed to methomyl oxime and carbon dioxide (Fig. 3D). This was different from the catabolism pathway previously proposed for the degradation of methomyl by strain MDW-2. In that pathway, methomyl was hydrolyzed to methomyl oxime and methylcarbamic acid (27) (Fig. S1), as only methomyl oxime was detected during the analysis of degrading product of methomyl by strain MDW-2, while HPLC did not detect methylcarbamic acid. Moreover, methylcarbamic acid could not be used for the growth and analytical experiments, as it is commercially unavailable. Therefore, this pathway was proposed based on the difference in chemical formulas of methomyl and methomyl oxime, in the absence of evidence of enzymes and genes. However, the results of the present study demonstrated the degradation of methomyl at the enzyme and gene levels and also corrected the shortcomings of the metabolic pathway of methomyl proposed by Zhang et al. (27). In this study, the hydrolyzing product of methomyl by AmeH was also the only one detected. Therefore, it was proposed that AmeH hydrolyzed methomyl to methylamine and an unstable compound that spontaneously transformed to methomyl oxime and carbon dioxide. The basis for this speculation is that AmeH is a carbamate C-N hydrolase from the FmdA_AmdA superfamily which catalyzes the cleavage of the amide bond; methomyl has only one amide bond. After being hydrolyzed, it forms methylamine and an unstable compound. Although methylamine was not detected, since strain MDW-2 was able to grow with methomyl as the sole carbon source, instead of methomyl oxime (27), this strain might use either methylamine or carbon dioxide as the sole carbon source for growth. However, strain MDW-2 can only use methylamine as the sole carbon to support its growth (Fig. S12), which also provides evidence for the speculation that methylamine was formed during the hydrolysis of methomyl. Accumulating studies have reported the generation of methylamine and carbon dioxide in the detoxification hydrolysis step (14,17,18). Knockout and complementation experiments with ameH have also proved that it is responsible for the hydrolysis of methomyl in strain MDW-2 (Fig. S9). The above information was combined to derive the metabolic pathway of methomyl in strain MDW-2 proposed here (Fig. S13). The unstable compound in the metabolic pathway was proposed based on the detection of methomyl oxime and the function of AmeH. In order to be strictly accurate, the unstable compound is indicated by square brackets in Fig. 3D.
Many amidases have been reported to catalyze the degradation of xenobiotics through the hydrolysis of C-N bonds (34)(35)(36)(37)(38)(39)(40)(41), such as hydrolysis of propham by the amidase MmH (37), hydrolysis of iprodione by the amidase IpaH (38), hydrolysis of N-methylpyrrolidone by an N-methylhydantoin amidohydrolase NmpAB (40), and hydrolysis of N,N-dimethylformamide by the dimethylformamidase DmfA1A2 (41). Most of these enzymes belong to the AS (amidase signature) family and often have a broad substrate spectrum (42). In comparison, only a few enzymes belong to the Fm-dA_AmdA superfamily, which often has a narrow substrate spectrum, limited to its specific substrate and simple amidase substrates only, such as formamide and acetamide (43,44). Therefore, the characteristics and catalytic mechanisms of these enzymes are less studied. In the present study, AmeH was classified as a member of the FmdA_AmdA superfamily (Fig. 2). Similar to other members of this superfamily, it has a narrow substrate spectrum. Among the tested substrates, apart from methomyl, AmeH could hydrolyze only formamide and acetamide. Five conserved amino acids (G162, N164, D191, H193, and E207) were identified as the key residues of AmeH (Table  S4), but whether these amino acid sites affect its correct folding or the active center requires further study on its crystal structure. Strains, plasmids, and culture conditions. The bacterial strains and plasmids used in this study are listed in Table 1, and the primers used are listed in Table 2. Strain MDW-2, a methomyl-degrading strain utilizing methomyl as the sole carbon source for growth, was previously isolated by our lab. It was cultured aerobically in LB broth or MSM at 30°C. Escherichia coli strains were cultured in LB broth on a rotary shaker (180 rpm) or on LB agar (1.5% [wt/vol]) plates at 37°C. Antibiotics were used at the following concentrations: ampicillin (Amp), 100 g ml Ϫ1 ; kanamycin (Km), 50 g ml Ϫ1 ; and tetracycline (Tc), 10 g ml Ϫ1 .
Purification of methomyl hydrolase. First, strain MDW-2 cells were cultured in LB broth, harvested by centrifugation at 6,000 ϫ g for 5 min at 4°C, and then washed twice with 20 mM Tris-HCl buffer (pH 7.5). The cell pellets were resuspended in 15 ml 20 mM Tris-HCl buffer (pH 7.5) and lysed by sonication (UH-650B ultrasonic processor; 40% intensity; Auto Science) in an ice bath for 15 min. Later, the lysate was centrifuged at 12,000 ϫ g for 30 min at 4°C, and the supernatant, i.e., the cell extract, was subjected to ammonium sulfate precipitation. The fraction between 40% and 60% ammonium sulfate was then collected by centrifugation at 12,000 ϫ g for 30 min at 4°C. The precipitate obtained was dissolved in 20 mM Tris-HCl buffer (pH 7.5) and dialyzed overnight in a Slide-A-Lyzer dialysis membrane (10 kDa) (Pierce, USA) against 20 mM Tris-HCl buffer (pH 7.5) at 4°C. After dialysis, the resulting mixture was subjected to DEAE-Sepharose fast-flow anion-exchange chromatography, and proteins were eluted with a 0-to-0.35 M linear gradient of NaCl in 20 mM Tris-HCl buffer (pH 7.5). The active fractions obtained were applied to a Q-Sepharose fast flow anion-exchange chromatography column and washed with a 0-to-0.5 M linear gradient of NaCl in 20 mM Tris-HCl buffer (pH 7.5). The active fractions were concentrated by Microcon centrifugal filters (10-kDa cutoff) and subjected to Sephadex-200 gel chromatography. The flow rate was 0.4 ml min Ϫ1 with 0.1 M NaCl in 20 mM Tris-HCl buffer (pH 7.5). The active fractions were concentrated with Microcon centrifugal filters (10-kDa cutoff). ExpressPlus PAGE gels (4 to 20%) purchased from GenScript (Nanjing) Co., Ltd., were employed to determine the molecular weight of the denatured protein (54). The protein components obtained from each purification step were subjected to a methomyl catalysis assay, and the component showing catalytic activity was named the active fraction. Protein concentrations were estimated by a Bradford assay (55).
Protein assay, sequencing, mass spectroscopy analysis, and genome comparison. The protein strip from the ExpressPlus PAGE gel (Fig. S2, lane 6) was excised and sent to Bo-Yuan Biological Technology Co., Ltd. (Shanghai, China), for peptide mass fingerprint analysis. The resulting peptide fragments were compared with the amino acid sequences of the annotated ORF from the draft genome of strain MDW-2 to identify high-identity sequences. Later, the related protein sequences were aligned  in Clustal X (version 2.1), and the phylogenetic tree was constructed by MEGA software (version 7.0) using the neighbor-joining method for phylogenetic analysis of methomyl hydrolase (AmeH) (56)(57)(58). The distances were calculated using a Kimura two-parameter distance model (59). Expression of ameH and purification of recombinant AmeH. ameH was amplified from the genomic DNA of strain MDW-2 using primers ameH-F and ameH-R ( Table 2). The resulting amplicon was ligated into the NdeI and XhoI sites of pET-24b(ϩ) using a ClonExpress II one-step cloning kit (Vazyme Biotech Co., Ltd., China) to produce pET-ameH, which was then transformed into E. coli BL21(DE3). The cells were cultured at an optical density at 600 nm (OD 600 ) of 0.5 in an LB medium supplemented with kanamycin (50 mg liter Ϫ1 ) at 37°C. Isopropyl-␤-D-thiogalactopyranoside (IPTG) was then added to a final concentration of 0.1 mM. The cells were incubated for an additional 12 h at 16°C, harvested by centrifugation, and subjected to ultrasonic disruption (UH-650B ultrasonic processor; 40% intensity; Auto Science) for 10 min. The lysate was then clarified by centrifugation at 12,000 ϫ g for 30 min to remove the intact cells (4°C). A nickel-nitrilotriacetic acid (Ni 2ϩ -NTA) resin was used to purify the enzymes from the supernatant (60). A series of imidazole concentrations were used to elute the recombinant AmeH from the resin. SDS-PAGE and Bradford assays were employed to determine the molecular weight and protein concentration, respectively (55). Gel filtration chromatography was used to determine the native molecular mass of AmeH. All the experiments were performed at a flow rate of 0.4 ml min Ϫ1 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 AmeH was estimated by plotting a calibration curve using 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).
Enzyme activity assay. The enzymatic reaction was performed at 30°C for 10 min in 1 ml of 20 mM Tris-HCl buffer (pH 7.5) containing 0.5 mM methomyl and 3 g AmeH. One enzyme activity unit was defined as the amount of enzyme required to hydrolyze 1 ⌴ methomyl per minute.
Enzyme kinetics were studied using various concentrations of methomyl (10 to 150 M) in the reaction mixture. The enzyme was diluted to 3 g to ensure that the substrate's consumption remained within the linear range during the reaction. The substrate concentration was determined based on the integration of chromatographic peak areas observed during HPLC analysis. K m and k cat values were calculated by nonlinear regression fitted to the Michaelis-Menten equation. All the reactions were carried out in triplicate, and the data were reported as means and standard deviations (SD).
Biochemical properties of recombinant AmeH. The concentrations of AmeH and methomyl used to investigate the optimal temperature and pH of AmeH were those used in the standard enzyme reaction. AmeH activity was tested between 15°C and 70°C (in increments of 5°C) to determine the optimal reaction temperature. The optimal reaction pH was assessed using several buffers with various pH values adjusted using 20 mM disodium hydrogen phosphate-citric acid buffer (pH 4.0 to 7.0), 20 mM Tris-HCl (pH 7.0 to 9.0), or 20 mM glycine-NaOH buffer (pH 9.0 to 10.0) (in increments of 0.5 pH unit). The thermal stability of AmeH was assessed by incubating the enzyme preparations at different temperatures for 1 h and measuring their residual activities under the assay conditions described above. The nonheated enzyme was used as the control (100% activity). AmeH was incubated at 4°C for 1 h in buffers with different pH values, and the residual activity was measured to determine pH stability. The samples were collected before the methomyl was completely consumed. The activity observed for the standard enzyme was defined as 100%, and the relative activities for individual reactions were calculated by comparing with the standard enzyme activity.
The AmeH substrate specificity was determined using carbaryl, isoprocarb, fenobucarb, propoxur, aldicarb, carbofuran, oxamyl, formamide, and acetamide. The assays were conducted under standard reaction conditions, as outlined above, with a 0.5 mM concentration of each individual substrate.
Construction of strain MDW-2M carrying an ameH gene disruption. A 500-bp DNA fragment (in the middle of ameH) was amplified from the genomic DNA of strain MDW-2 with pEX-F and pEX-R primers to disrupt ameH through a single crossover (61) ( Table 2). The fragment was cloned into plasmid pEX18Tc (62), digested with SacI and EcoRI using the ClonExpress II one-step cloning kit to generate pEX-ameH. pEX-ameH was introduced into strain MDW-2 by electroporation, as described by Zhang et al. (63). Single-crossover clones were selected on LB plates supplemented with kanamycin (50 g/ml) and tetracycline (10 g/ml). The ameH disruption mutant, designated strain MDW-2M, was verified by PCR. The loss of its ability to degrade methomyl was tested in MSM supplemented with 0.5 mM methomyl.
Complementation of the ameH disruption mutant. A 951-bp fragment of ameH was PCR amplified from the genomic DNA of strain MDW-2 using primers pBBR1-F and pBBR1-R ( Table 2). The PCR product was cloned into EcoRI and BamHI sites of the broad-host-range plasmid pBBR1MCS-4 (64) using the ClonExpress II one-step cloning kit to generate pBBR1-ameH. This plasmid was introduced into strain MDW-2M by electroporation to generate strain MDW-2M (pBBR1-ameH). The ability of this strain to degrade methomyl was tested in MSM supplemented with 0.5 mM methomyl.
Site-directed mutagenesis. The point mutations were introduced in the ameH gene by overlap PCR. ameH-F and ameH-R were used as the forward and reverse flanking primers, respectively. The internal primer pairs G162A-F/R, N164A-F/R, D191A-F/R, H193A-F/R, and E207A-F/R are listed in Table 2. All PCRs were performed using Phanta Max Super-Fidelity DNA polymerase (Vazyme Biotech Co., Ltd., China) with the standard site-directed mutagenesis protocol (65). The PCR products were gel purified and cloned into NdeI and XhoI sites of pET-24b(ϩ), as described above. Successful substitutions were confirmed by DNA sequencing. Purification of the recombinant proteins and their activity were detected, as described above.