Identification of anopd(Organophosphate Degradation) Gene in anAgrobacteriumIsolate

Abstract

We isolated a bacterial strain, Agrobacterium radiobacter P230, which can hydrolyze a wide range of organophosphate (OP) insecticides. A gene encoding a protein involved in OP hydrolysis was cloned from A. radiobacter P230 and sequenced. This gene (called opdA) had sequence similarity to opd, a gene previously shown to encode an OP-hydrolyzing enzyme in Flavobacterium sp. strain ATCC 27551 and Brevundimonas diminuta MG. Insertional mutation of the opdA gene produced a strain lacking the ability to hydrolyze OPs, suggesting that this is the only gene encoding an OP-hydrolyzing enzyme in A. radiobacter P230. The OPH and OpdA proteins, encoded by opd and opdA, respectively, were overexpressed and purified as maltose-binding proteins, and the maltose-binding protein moiety was cleaved and removed. Neither protein was able to hydrolyze the aliphatic OP malathion. The kinetics of the two proteins for diethyl OPs were comparable. For dimethyl OPs, OpdA had a higher k cat than OPH. It was also capable of hydrolyzing the dimethyl OPs phosmet and fenthion, which were not hydrolyzed at detectable levels by OPH.Synthetic organophosphates (OPs) are used widely as insecticides in agriculture. OPs contain three phosphoester linkages and are hence often termed phosphotriesters. The phosphorus is also linked by a double bond to either an oxygen (PAO) in oxon OPs or a sulfur (PAS) in thion OPs. These insecticides are potent acetylcholinesterase (AchE) inhibitors, and various clinical effects can occur due to OP poisoning in humans. In general, hydrolysis of one of the phosphoester bonds reduces the toxicity of an OP, and in the case of parathion (O,O-diethyl p-nitrophenyl phosphorothioate), a 100-fold reduction in toxicity occurs (37,38).Enzymatic detoxification of OPs has become the focus of many studies because other means of removing OP residues are impractical or costly or are themselves environmentally hazardous. Enzymes from insect species resistant to OPs have been identified and considered for use in bioremediation (27). However, these enzymes are capable of hydrolyzing only oxon OPs and function at rates several orders of magnitude below the diffusion-limited maximum rate (28). Bacterial enzymes have also received considerable attention and may have advantages in terms of broader substrate specificities (both oxon and thion OPs) and superior kinetics (10, 13). The most widely studied bacterial enzyme is the OPH (organophosphorus-hydrolyzing) protein.OPH is a zinc-containing homodimeric protein found in the membrane of Flavobacterium sp. strain ATCC 27551 and Brevundimonas diminuta MG (5, 13). OPH is capable of hydrolyzing a wide range of oxon and thion OPs (13) and hydrolyzes paraoxon at a rate approaching the diffusion limits (6, 35). The OPH enzyme is encoded by the opd gene. Other opd-containing organisms (for example, a Pseudomonas strain) have been identified by using this gene in Southern hybridization analysis (8), while other OP-hydrolyzing organisms clearly do not contain the opd gene (10, 11). Flavobacterium sp...

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].…”

“…The enzyme is encoded by an opd (organophosphate degradation) gene and is capable of hydrolyzing a wide range of oxon and thion organophosphates [20,21]. The opd gene has been detected in soil microorganisms belonging to different taxonomic groups, which points to its horizontal spread using mobile genetic elements (transposons, plasmids, phages) as transfer vehicles [22][23][24][25][26][27][28].…”

Spectroscopic and mechanistic studies of dinuclear metallohydrolases and their biomimetic complexes

“…more solvent exposed 6-coordinate β-site. 49,[68][69][70][71] The metals are bridged by a carboxylated lysine residue in addition to a µ-hydroxide. The active site of OpdA is shown in Fig.…”

The Art of Immobilization Using Biopolymers, Biomaterials and Nanobiotechnology

“…One of the most important technologies to be applied for this approach is immobilized enzyme. The immobilized enzyme is capable of breaking down a range of pesticide-contaminated waste as organophosphate insecticides (Horne et al, 2002;Sharmin et al, 2007).…”