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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.

SUBMITTER: Jiang W

PROVIDER: S-EPMC7755249 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Carbofuran, a broad-spectrum systemic insecticide, has been extensively used for approximately 50 years. Diverse carbofuran-degrading bacteria have been described, among which sphingomonads have exhibited an extraordinary ability to catabolize carbofuran; other bacteria can only convert carbofuran to carbofuran phenol, while all carbofuran-degrading sphingomonads can degrade both carbofuran and carbofuran phenol. However, the genetic basis of carbofuran catabolism in sphingomonads has not been well elucidated. In this work, we sequenced the draft genome of Sphingomonas sp. strain CDS-1 that can transform both carbofuran and carbofuran phenol but fails to grow on them. On the basis of the hypothesis that the genes involved in carbofuran catabolism are highly conserved among carbofuran-degrading sphingomonads, two such genes, cehACDS-1 and cfdCCDS-1, were predicted from the 84 open reading frames (ORFs) that share ?95% nucleic acid similarities between strain CDS-1 and another sphingomonad Novosphingobium sp. strain KN65.2 that is able to mineralize the benzene ring of carbofuran. The results of the gene knockout, genetic complementation, heterologous expression, and enzymatic experiments reveal that cehACDS-1 and cfdCCDS-1 are responsible for the conversion of carbofuran and carbofuran phenol, respectively, in strain CDS-1. CehACDS-1 hydrolyzes carbofuran to carbofuran phenol. CfdCCDS-1, a reduced flavin mononucleotide (FMNH2)- or reduced flavin adenine dinucleotide (FADH2)-dependent monooxygenase, hydroxylates carbofuran phenol at the benzene ring in the presence of NADH, FMN/FAD, and the reductase CfdX. It is worth noting that we found that carbaryl hydrolase CehAAC100, which was previously demonstrated to have no activity toward carbofuran, can actually convert carbofuran to carbofuran phenol, albeit with very low activity.IMPORTANCE Due to the extensive use of carbofuran over the past 50 years, bacteria have evolved catabolic pathways to mineralize this insecticide, which plays an important role in eliminating carbofuran residue in the environment. This study revealed the genetic determinants of carbofuran degradation in Sphingomonas sp. strain CDS-1. We speculate that the close homologues cehA and cfdC are highly conserved among other carbofuran-degrading sphingomonads and play the same roles as those described here. These findings deepen our understanding of the microbial degradation mechanism of carbofuran and lay a foundation for the better use of microbes to remediate carbofuran contamination.

Project description:Iprodione [3-(3,5-dichlorophenyl) N-isopropyl-2,4-dioxoimidazolidine-1-carboxamide] is a highly effective broad-spectrum dicarboxamide fungicide. Several bacteria with iprodione-degrading capabilities have been reported; however, the enzymes and genes involved in this process have not been characterized. In this study, an iprodione-degrading strain, Paenarthrobacter sp. strain YJN-5, was isolated and characterized. Strain YJN-5 degraded iprodione through the typical pathway, with hydrolysis of its N-1 amide bond to N-(3,5-dichlorophenyl)-2,4-dioxoimidazolidine as the initial step. The ipaH gene, encoding a novel amidase responsible for this step, was cloned from strain YJN-5 by the shotgun method. IpaH shares the highest similarity (40%) with an indoleacetamide hydrolase (IAHH) from Bradyrhizobium diazoefficiens USDA 110. IpaH displayed maximal enzymatic activity at 35°C and pH 7.5, and it was not a metalloamidase. The kcat and Km of IpaH against iprodione were 22.42 s-1 and 7.33 ?M, respectively, and the catalytic efficiency value (kcat/Km ) was 3.09 ?M-1 s-1 IpaH has a Ser-Ser-Lys motif, which is conserved among members of the amidase signature family. The replacement of Lys82, Ser157, and Ser181 with alanine in IpaH led to the complete loss of enzymatic activity. Furthermore, strain YJN-5M lost the ability to degrade iprodione, suggesting that ipaH is the only gene responsible for the initial iprodione degradation step. The ipaH gene could also be amplified from another previously reported iprodione-degrading strain, Microbacterium sp. strain YJN-G. The sequence similarity between the two IpaHs at the amino acid level was 98%, indicating that conservation of IpaH exists in different strains.IMPORTANCE Iprodione is a widely used dicarboxamide fungicide, and its residue has been frequently detected in the environment. The U.S. Environmental Protection Agency has classified iprodione as moderately toxic to small animals and a probable carcinogen to humans. Bacterial degradation of iprodione has been widely investigated. Previous studies demonstrate that hydrolysis of its N-1 amide bond is the initial step in the typical bacterial degradation pathway of iprodione; however, enzymes or genes involved in iprodione degradation have yet to be reported. In this study, a novel ipaH gene encoding an amidase responsible for the initial degradation step of iprodione in Paenarthrobacter sp. strain YJN-5 was cloned. In addition, the characteristics and key amino acid sites of IpaH were investigated. These findings enhance our understanding of the microbial degradation mechanism of iprodione.

Project description:Strobilurin fungicides are widely used in agricultural production due to their broad-spectrum and fungal mitochondrial inhibitory activities. However, their massive application has restrained the growth of eukaryotic algae and increased collateral damage in freshwater systems, notably harmful cyanobacterial blooms (HCBs). In this study, a strobilurin fungicide-degrading strain, Hyphomicrobium sp. strain DY-1, was isolated and characterized successfully. Moreover, a novel esterase gene, strH, responsible for the de-esterification of strobilurin fungicides, was cloned, and the enzymatic properties of StrH were studied. For trifloxystrobin, StrH displayed maximum activity at 50°C and pH 7.0. The catalytic efficiencies (kcat/Km ) of StrH for different strobilurin fungicides were 196.32 ± 2.30 μM-1 · s-1 (trifloxystrobin), 4.64 ± 0.05 μM-1 · s-1 (picoxystrobin), 2.94 ± 0.02 μM-1 · s-1 (pyraclostrobin), and (2.41 ± 0.19)×10-2 μM-1 · s-1 (azoxystrobin). StrH catalyzed the de-esterification of a variety of strobilurin fungicides, generating the corresponding parent acid to achieve the detoxification of strobilurin fungicides and relieve strobilurin fungicide growth inhibition of Chlorella This research will provide insight into the microbial remediation of strobilurin fungicide-contaminated environments.IMPORTANCE Strobilurin fungicides have been widely acknowledged as an essential group of pesticides worldwide. So far, their residues and toxic effects on aquatic organisms have been reported in different parts of the world. Microbial degradation can eliminate xenobiotics from the environment. Therefore, the degradation of strobilurin fungicides by microorganisms has also been reported. However, little is known about the involvement of enzymes or genes in strobilurin fungicide degradation. In this study, a novel esterase gene responsible for the detoxification of strobilurin fungicides, strH, was cloned in the newly isolated strain Hyphomicrobium sp. DY-1. This degradation process detoxifies the strobilurin fungicides and relieves their growth inhibition of Chlorella.

Project description:Pendimethalin [N-(1-ethylpropyl)-2,6-dinitro-3,4-xylidine] is a selective preemergence dinitroaniline herbicide. Several fungi and bacteria have been reported to degrade pendimethalin, but the enzymes or genes involved in this process have not been characterized. Nitroreduction is the initial degradation and detoxification step for pendimethalin. In this study, a pendimethalin nitroreductase (PNR), responsible for the nitroreduction of pendimethalin, was purified from the pendimethalin-degrading strain Bacillus subtilis Y3. Based on a comparison of its mass fingerprints with all of the deduced proteins from the draft genome of strain Y3, a protein annotated as a nitroreductase was identified, and its corresponding encoding gene was termed pnr PNR was a functional homodimer with a subunit molecular mass of approximately 23 kDa. PNR reduced the C-6 nitro group of the aromatic ring of pendimethalin, yielding 2-nitro-6-amino-N-(1-ethylpropyl)-3,4-xylidine. PNR could also catalyze the nitroreduction of three other major varieties of dinitroaniline herbicides, including butralin, oryzalin, and trifluralin. However, the number of reduced nitro groups was two instead of one, which differed from the nitroreduction of pendimethalin by PNR and which may be due to the symmetry in the chemical structures of the two nitro groups. A detoxification assay revealed that 2-nitro-6-amino-N-(1-ethylpropyl)-3,4-xylidine (PNR-reduced pendimethalin) showed no inhibitory effect on the growth of Saccharomyces cerevisiae BY4741, whereas pendimethalin showed an obvious inhibitory effect on its growth, indicating the detoxification effect of pendimethalin by PNR. Therefore, PNR has potential in pendimethalin detoxification applications. This report describes an enzyme (and corresponding gene) involved in the biodegradation of pendimethalin and dinitroaniline herbicides.Pendimethalin [N-(1-ethylpropyl)-2,6-dinitro-3,4-xylidine] is a widely used selective preemergence dinitroaniline herbicide, and its residue has been frequently detected in the environment. The U.S. Environmental Protection Agency (EPA) has classified pendimethalin as a persistent bioaccumulative toxin. To date, no enzymes or genes involved in pendimethalin biodegradation have been reported. In the present study, the gene pnr, which encodes the nitroreductase PNR, responsible for the nitroreduction of pendimethalin, was cloned from the pendimethalin-degrading strain Bacillus subtilis Y3. PNR could also catalyze the nitroreduction of three other major varieties of dinitroaniline herbicides, including butralin, oryzalin, and trifluralin. The reduction of pendimethalin by PNR might eliminate its toxicity against Saccharomyces cerevisiae BY4741, indicating the application potential of PNR in the detoxification of pendimethalin.

Project description:A Gram-positive rubber-degrading bacterium, Actinoplanes sp. strain OR16 (strain NBRC 114529), is able to grow on agar plates containing natural and synthetic rubber as the sole sources of carbon and energy. When this strain was grown on natural rubber latex overlay agar plates, translucent halos around the cells were observed. To identify the natural rubber degradation genes and other features of its metabolism, its complete genome sequence was determined. The genome of OR16 consists of 9,293,892 bp and comprises one circular chromosome (GenBank accession number AP019371.1) with a G + C content of 70.3%. The genome contains 8238 protein-coding and 18 rRNA genes. A homology search of the genome sequence revealed that three genes (lcp1, lcp2, and lcp3) are homologous to an extracellular latex-clearing protein (Lcp) of Streptomyces sp. K30. RT-PCR analysis revealed that lcp1 and lcp2 seem to constitute an operon. Purified lcp gene products have oxygen consumption activity toward natural rubber latex, suggesting that all these genes encode rubber-degrading enzymes in OR16. Quantitative reverse transcription-PCR analysis indicated that the transcription of these genes is induced during the growth of OR16 on natural rubber. The genes located adjacent to lcp1 and lcp3, which code for a TetR/AcrR-type transcriptional regulator, can bind to the promoter regions of these lcp genes. It is suggested that the putative regulators play a role in regulating the transcription of the lcp genes. These results strongly suggested that three lcp genes are required for the utilization of natural rubber in strain OR16. Key Points • The complete genome sequence of Actinoplanes sp. strain OR16 was determined. • Three lcp genes which are involved in the natural rubber degradation in OR16 were identified. • Transcription of these lcp genes is induced during utilization of rubber in OR16. • Two regulators, which bind to the promoter regions of lcp, were determined.

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

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