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

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. K_m and k_cat of AmeH for methomyl were 87.5 μM and 345.2 s^-1, respectively, and catalytic efficiency (k_cat/K_m ) 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.

Keywords: Aminobacter aminovorans MDW-2; biodegradation; carbamate C-N hydrolase; methomyl.

FIG 1

Chemical structure of the carbamate insecticides.

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 >50% 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, Paracoccidioides brasiliensis ForM; AAG60585, Aspergillus nidulans ForM; Q9URY7, Schizosaccharomyces pombe ForM; ACM68705, Lupinus albus ForM; Q50228, Methylophilus methylotrophus ForM; Q07838, Mycobacterium smegmatis ForM; AAM25099, Thermoanaerobacter tengcongensis AceM; WP044436960, Skermanella aerolata AceM; WP013640752, Acidiphilium multivorum AceM; WP039888139, Acidiphilium sp. PM AceM; WP057592521, Variovorax paradoxus AceM; WP012764433, Dickeya paradisiaca AceM; WP034389513, Comamonas thiooxydans AceM; WP018421042, Burkholderiaceae AceM. In the amidase signature family clan, accession numbers are as follows: CAG29798, Microbacterium sp. AJ115 Ami; D16207, Rhodococcus rhodochrous J1 Ami; AF290611, Sulfolobus solfataricus Ami; AAK11724, Rhodococcus erythropolis MP50 Ami; AAS87173, Achromobacter xylosoxidans Ami; CAC93616, Stenotrophomonas maltophilia Ami; ALB08735, Delftia tsuruhatensis Ami; ALB08736, Delftia tsuruhatensis Ami; AAD01507, Bradyrhizobium japonicum Ami. In the nitrilase superfamily clan, accession numbers are as follows: XP002179811, Phaeodactylum tricornutum CCAP 1055/1 ForM; NP791184, Pseudomonas syringae ALAM; WP011654378, Rhizobium leguminosarum ForM; O25836, Helicobacter pylori ForM; ADQ27473, Bacillus cereus CECT 5050T ForM; CAA72932, Helicobacter pylori ALAM; WP012745300, Variovorax paradoxus ALAM; AAX83004, Rhodococcus rhodochrous ALAM; AAF14257, Bacillus stearothermophilus BR388 Ami; AAA22990, Brevibacterium sp. R312 Ami; WP012723740, Pseudomonas fluorescens ALAM.

FIG 3

HPLC and MS/MS analyses of the products of methomyl treated with purified AmeH. (A) HPLC analysis of metabolites that appeared during the conversion of methomyl by AmeH. (B) MS/MS analysis of compound I (m/z 163.0534 [M+H]⁺), identified as methomyl. (C) MS/MS analysis of compound II (m/z 106.0322 [M+H]⁺), identified as methomyl oxime. (D) The conversion pathway of methomyl by AmeH.