Parathion hydrolase specified by the Flavobacterium opd gene: relationship between the gene and protein

Abstract

The sequence of a 1,693-base-pair plasmid DNA fragment from Flavobacterium sp. strain ATCC 27551 containing the parathion hydrolase gene (opd) was determined. Within this sequence, there is only one open reading frame large enough to encode the 35,000-dalton membrane-associated hydrolase protein purified from Flavobacterium extracts. Amino-terminal sequence analysis of the purified Flavobacterium hydrolase demonstrated that serine is the amino-terminal residue of the hydrolase protein. The amino-terminal serine corresponds to a TCG codon located 87 base pairs downstream of the presumptive ATG initiation codon in the nucleotide sequence. The amino acid composition of the purified protein agrees well with that predicted from the nucleotide sequence, using serine as the amino-terminal residue. These data suggest that the parathion hydrolase protein is processed at its amino terminus in Flavobacterium sp. Construction in Escherichia coli of a lacZ-opd gene fusion in which the first 33 amino-terminal residues of opd were replaced by the first 5 residues of lacZ resulted in the production of an active hydrolase identical in molecular mass to the hydrolase isolated from Flavobacterium sp. E. coli cells containing the lacZ-opd fusion showed higher levels of hydrolase activity than did cells containing the parent plasmid.The microbial degradation of hazardous waste offers a promising strategy by which such some wastes may be economically and safely detoxified. For selected situations, microbial processes have considerable advantages over other technologies in that microbial processes can yield precise products, function at low concentrations of solute, and require relatively low levels of technology for construction and maintenance. However, there are relatively few instances in which microbial processes are being actively used to control hazardous wastes.Organophosphate compounds such as the insecticide parathion (O,O-diethyl-0-4-nitrophenyl phosphorothioate) are susceptible to microbial hydrolysis by bacterial parathion hydrolases. In fact, such soil hydrolases are thought to play a role in the relatively low persistence of these compounds. Since organophosphates constitute a large fraction of the insecticides used in the industrialized countries, there is a need for economical and reliable methods to detoxify organophosphate wastes (such as residual pesticide concentrates in their original containers, contaminated stock solutions, and dilute pesticide solutions resulting from the washing of spraying equipment). This need has stimulated recent research on parathion hydrolases.Parathion hydrolase activities have been described in a variety of bacterial isolates and are characterized by broad substrate ranges for compounds structurally related to parathion, broad temperature and pH optima, and high stability (1,10,13,14). Of the parathion hydrolases that have been characterized in detail, the enzymes encoded by the related opd genes of Flavobacterium sp. strain ATCC 27551 and Pseudomonas diminuta MG are noteworthy becaus...

Cross-linked enzyme crystals of organophosphate hydrolase for electrochemical detection of organophosphorus compounds

“…Recombinant expression of OPH usually results in low active enzyme yield due to low solubility of this protein in E. coli (Mulbry and Karns 1989a;Grimsley et al 1997). Though several approaches have been investigated for improved production of OPH in recombinant systems, including multi-gene fusions (Wu et al 2001), fusion with highly-soluble fusion partners (Cha et al 2000), display on cell surface (Richins et al 1997;Li et al 2004), and periplasmic secretion (Kang et al 2005), reaching high productivity of OPH using E. coli-based fermentation or in other recombinant systems (Takayama et al 2006) is still challenging, especially for further cost-efficient production of highly purified enzymes for electrochemical biosensor applications.…”

Isolation and molecular characterization of a novel <i>pseudomonas putida</i> strain capable of degrading organophosphate and aromatic compounds

“…Therefore, we attempted to identify organophosphorus hydrolase gene by PCR. Among OPhydrolyzing enzymes, the most studied so far is a product of opd gene, which was found in Sphingobium fuliginis [27,59], Brevundimonas diminuta GM [17,27], and in several representatives of genera Pseudomonas and Agrobacterium (in the latter case the gene is designnated opdA) [24][25][26]60]. Detection of the gene in the POXN01 strain was performed according to an established protocol that relies on analytical PCR with opdspecific primers [32].…”

Microbial metabolism of sulfurand phosphorus-containing xenobiotics

“…ATCC 27551 [62], and from two unidentified strains, B-1 and SC [56]. In the former two organisms, it is a 35-kDa, membrane-associated enzyme, and in P. diminuta it appears to exist as a dimer [61].…”

“…ATCC 27551 have both been shown to be encoded on large plasmids, pCMS1 (51 kb) and pPDL2 (39 kb (also called pSM55; 43 kb [64]), respectively [61,62]. The two opd (organophosphate degradation) genes have been cloned and sequenced [61,62,65], and proved to be 100% identical [64], although restriction mapping confirmed that the plasmids carrying them differed extensively [64]. Both of these enzymes are produced constitutively, and have usually been purified from nutrient broth cultures grown without addition of phosphotriesters as inducers.…”