Molecular cloning and nucleotide sequencing of organophosphorus insecticide hydrolase gene from arthrobacter sp. strain B-5

Ohshiro, Kazufumi; Kakuta, Takasuke; Nikaidou, Naoki; Watanabe, Takeshi; Uchiyama, Takeo

doi:10.1016/s1389-1723(99)80105-4

Cited by 29 publications

(22 citation statements)

References 13 publications

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“…A. globiformis D47 is a member of the gram-positive coryneform bacteria, and therefore it is surprising that a promoter from this strain can function in E. coli. However a few A. globiformis genes have been expressed in E. coli (2,4,5,10,20,22,27), and some are also known to be expressed from their own promoters (27). Identification of the degradative gene(s) and sequence analysis.…”

Section: Resultsmentioning

confidence: 99%

Degradation of Substituted Phenylurea Herbicides by Arthrobacter globiformis Strain D47 and Characterization of a Plasmid-Associated Hydrolase Gene, puhA

Turnbull¹,

Ousley²,

Walker³

et al. 2001

Appl Environ Microbiol

106

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Arthrobacter globiformis D47 was shown to degrade a range of substituted phenylurea herbicides in soil. This strain contained two plasmids of approximately 47 kb (pHRIM620) and 34 kb (pHRIM621). Plasmid-curing experiments produced plasmid-free strains as well as strains containing either the 47-or the 34-kb plasmid. The strains were tested for their ability to degrade diuron, which demonstrated that the degradative genes were located on the 47-kb plasmid. Studies on the growth of these strains indicated that the ability to degrade diuron did not offer a selective advantage to A. globiformis D47 on minimal medium designed to contain the herbicide as a sole carbon source. The location of the genes on a plasmid and a lack of selection would explain why the degradative phenotype, as with many other pesticide-degrading bacteria, can be lost on subculture. A 22-kb EcoRI fragment of plasmid pHRIM620 was expressed in Escherichia coli and enabled cells to degrade diuron. Transposon mutagenesis of this fragment identified one open reading frame that was essential for enzyme activity. A smaller subclone of this gene (2.5 kb) expressed in E. coli coded for the protein that degraded diuron. This gene and its predicted protein sequence showed only a low level of protein identity (25% over ca. 440 amino acids) to other database sequences and was named after the enzyme it encoded, phenylurea hydrolase (puhA gene).

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Section: Resultsmentioning

confidence: 99%

Degradation of Substituted Phenylurea Herbicides by Arthrobacter globiformis Strain D47 and Characterization of a Plasmid-Associated Hydrolase Gene, puhA

Turnbull¹,

Ousley²,

Walker³

et al. 2001

Appl Environ Microbiol

106

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“…strain B-5 (GenBank accession no. BAA85881), (23), or aryldialkylphosphatase from Nocardia sp. B-1 (GenBank accession no.…”

Section: Discussionmentioning

confidence: 99%

Gulosibacter molinativorax ON4 ^T Molinate Hydrolase, a Novel Cobalt-Dependent Amidohydrolase

et al. 2011

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A new pathway of molinate mineralization has recently been described. Among the five members of the mixed culture able to promote such a process, Gulosibacter molinativorax ON4 T has been observed to promote the initial breakdown of the herbicide into ethanethiol and azepane-1-carboxylate. In the current study, the gene encoding the enzyme responsible for molinate hydrolysis was identified and heterologously expressed, and the resultant active protein was purified and characterized. Nucleotide sequence analysis revealed that the gene encodes a 465-amino-acid protein of the metal-dependent hydrolase A subfamily of the amidohydrolase superfamily with a predicted molecular mass of 50.9 kDa. Molinate hydrolase shares the highest amino acid sequence identity (48 to 50%) with phenylurea hydrolases of Arthrobacter globiformis and Mycobacterium brisbanense. However, in contrast to previously described members of the metal-dependent hydrolase A subfamily, molinate hydrolase contains cobalt as the only active-site metal.Molinate is a herbicide which is extensively applied to rice fields worldwide. When this thiocarbamate herbicide is applied to the flooded paddies, it dissipates into the environment largely through volatilization. However, (photo)chemical and microbiological molinate transformation also occurs (28), resulting in accumulation of oxidized metabolites, such as oxomolinate and molinate sulfoxide (11, 13), which have increased toxicity (5). The only biological system described so far as being able to mineralize molinate and use the herbicide as the sole source of carbon, energy, and nitrogen is a five-membered bacterial mixed culture (2, 6). Among the five community members, Gulosibacter molinativorax ON4 T (gen. nov., sp. nov.), the only representative of this genus thus far (21), is responsible for the initial breakdown of molinate. In contrast to previously described molinate transformation reactions (13), G. molinativorax ON4T cleaves the thioester bond of molinate, releasing ethanethiol and azepane-1-carboxylate (ACA) (Fig. 1) (1). While this hydrolysis proceeds in the absence of oxygen, the further metabolism and mineralization of ACA by G. molinativorax ON4 T necessitate oxygen. Ethanethiol is not transformed by this organism but is spontaneously oxidized to diethyl disulfide. At molinate concentrations of Ͼ2 mM, the accumulating sulfur compounds are toxic for G. molinativorax ON4T and molinate mineralization is achieved only when other mixed-culture members able to degrade the sulfurcontaining metabolites are present (1, 2).In the present study, the molinate hydrolase (MolA) from G. molinativorax ON4T responsible for the key step of molinate breakdown was purified and characterized. The encoding gene was identified and expressed in Escherichia coli. This enzyme belongs to the metal-dependent hydrolase A subfamily of the amidohydrolase superfamily; however, in contrast to previously characterized members, it contains cobalt as a cofactor. To the best of our knowledge, this is the first description of an...

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“…strain scl-2 using the primer pair 12F/12R that was designed based on the sequence of the isofenphos hydrolase gene (Ohshiro et al 1999). The amplified fragment was subcloned into the linear vector pMD18-T and transformed into the competent E. coli DH5α.…”

Section: Resultsmentioning

confidence: 99%

“…strain B-5 in 1997 (Ohshiro et al 1997), which shares 58.1% identity with AdpB from Nocardia sp. strain B-1 (Ohshiro et al 1999;Ohshiro et al 1997). To the best of our knowledge, this hydrolase is the only enzyme reported to hydrolyze isofenphos.…”

Section: Introductionmentioning

confidence: 92%

An isofenphos-methyl hydrolase (Imh) capable of hydrolyzing the P–O–Z moiety of organophosphorus pesticides containing an aryl or heterocyclic group

Liu

Zhang

et al. 2011

Appl Microbiol Biotechnol

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Organophosphorus pesticide (OP) hydrolases play key roles in the degradation and decontamination of agricultural and household OPs and in the detoxification of chemical warfare agents. In this study, an isofenphos-methyl hydrolase gene (imh) was cloned from the isocarbophos-degrading strain of Arthrobacter sp. scl-2 using the polymerase chain reaction method. Isofenphos-methyl hydrolase (Imh) showed 98% sequence identity with the isofenphos hydrolase from Arthrobacter sp. strain B-5. Imh was highly expressed in Escherichia coli BL21 (DE3), and the His(6)-tagged Imh was purified (1.7 mg/ml) with a specific activity of 14.35 U/mg for the substrate isofenphos-methyl. The molecular mass of the denatured Imh is about 44 kDa, and the isoelectric point (pI) value was estimated to be 3.4. The optimal pH and temperature for hydrolysis of isofenphos-methyl were pH 8.0 and 35 °C, respectively. The secondary structure of Imh shows that Imh is a metallo-dependent hydrolase, and it was found that Imh was completely inhibited by the metalloprotease inhibitor 1,10-phenanthroline (0.5 mM), and the catalytic activity was restored by the subsequent addition of Zn(2+). Interestingly, Imh had a relatively broader substrate specificity and was capable of hydrolyzing 12 of the tested oxon and thion OPs with the P-O-Z moiety instead of the P-S(C)-Z moiety. Furthermore, it was found that the existence of an aryl or heterocyclic group in the leaving group (Z) is also important in determining the substrate specificity. Among all the substrates hydrolyzed by Imh, it was assumed that Imh preferred P-O-Z substrates still with a phosphamide bond (P-N), such as isofenphos-methyl, isofenphos, isocarbophos, and butamifos. The newly characterized Imh has a great potential for use in the decontamination and detoxification of agricultural and household OPs and is a good candidate for the study of the catalytic mechanism and substrate specificity of OP hydrolases.

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Molecular cloning and nucleotide sequencing of organophosphorus insecticide hydrolase gene from arthrobacter sp. strain B-5

Cited by 29 publications

References 13 publications

Degradation of Substituted Phenylurea Herbicides by Arthrobacter globiformis Strain D47 and Characterization of a Plasmid-Associated Hydrolase Gene, puhA

Degradation of Substituted Phenylurea Herbicides by Arthrobacter globiformis Strain D47 and Characterization of a Plasmid-Associated Hydrolase Gene, puhA

Gulosibacter molinativorax ON4 ^T Molinate Hydrolase, a Novel Cobalt-Dependent Amidohydrolase

An isofenphos-methyl hydrolase (Imh) capable of hydrolyzing the P–O–Z moiety of organophosphorus pesticides containing an aryl or heterocyclic group

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Molecular cloning and nucleotide sequencing of organophosphorus insecticide hydrolase gene from arthrobacter sp. strain B-5

Cited by 29 publications

References 13 publications

Degradation of Substituted Phenylurea Herbicides by Arthrobacter globiformis Strain D47 and Characterization of a Plasmid-Associated Hydrolase Gene, puhA

Degradation of Substituted Phenylurea Herbicides by Arthrobacter globiformis Strain D47 and Characterization of a Plasmid-Associated Hydrolase Gene, puhA

Gulosibacter molinativorax ON4 T Molinate Hydrolase, a Novel Cobalt-Dependent Amidohydrolase

An isofenphos-methyl hydrolase (Imh) capable of hydrolyzing the P–O–Z moiety of organophosphorus pesticides containing an aryl or heterocyclic group

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Product

Resources

About

Gulosibacter molinativorax ON4 ^T Molinate Hydrolase, a Novel Cobalt-Dependent Amidohydrolase