Aryldialkylphosphatase

Bacterial isolates capable of degrading organophosphate (OP) pesticides have been identified from soil samples from different parts of the world.[3][4] The first organophosphate-degrading bacterial species was isolated from a soil sample from the Philippines in 1973,[5] which identified as Flavobacterium sp. ATCC27551. Since then, other species have demonstrated to have OP-degrading abilities, such as Pseudomonas diminuta (isolated from US soil sample), Agrobacterium radiobacter (isolated from Australian soil sample), Alteromonas haloplanktis (isolated from US soil sample), and Pseudomonas sp. WBC-3 (isolated from Chinese soil sample).[3]

The capacity to hydrolyze organophosphates is not unique to bacteria. A few fungi and cyanobacteria species have been found to also hydrolyze them.[3] Moreover, through sequence homology searches of whole genomes, several other bacterial species were identified that also contain sequences from the same gene family as opd, including pathogenic bacteria such as Escherichia coli (yhfV) and Mycobacterium tuberculosis.[3]

The gene sequence encoding the enzyme (opd) in Flavobacterium sp. ATCC27551 and Pseudomonas diminuta is highly conserved (100% sequence homology),[4] although the plasmids where the genes are found have very different sequences apart from a 5.1Kb[4][6] conserved region where the gene is found.[2]

A closer look on the organization of the opd gene from Flavobacterium suggests a potential transposon-like architecture, which accounts for the widespread distribution of the gene among other microbial species that might have occurred through lateral DNA transfer. The opd gene is flanked by transposition insertion sequences, characteristic of Tn3 family of transposons. Moreover, a transposase-like sequence (homologous to TnpA) and a resolvase-like sequence (homologous to TnpR) were also identified in regions upstream of the opd gene,[4] which are characteristics of class II transposons such as Tn3.

Furthermore, another open reading frame was identified downstream of opd and encodes a protein that further degrades p-nitrophenol, one of the byproducts of OP degradation. This protein is believed to work as a complex with PTE, since a dramatic increase in activity is observed when PTE is present.[4]

Therefore, the characteristic architectural organization of the opd gene region suggests that different species acquired the gene through horizontal transfer through transposition and plasmid transfer.

Phosphotriesterase is present in two species, Pseudomonas diminuta and Flavobacterium sp. ATCC27551. Other gene variants that also encode organophosphate-degrading enzymes are present in other species. The list includes bacterial species such as the radioresistant Deinococcus radiodurans, pathogens Mycobacterium tuberculosis and Mycobacterium bovis, the anaerobic bacterium Desulfatibacillum alkenivorans, the thermophilic bacteria Geobacillus sp. and Thermoanaerobacter sp. X514, Escherichia coli (yhfV) and many other groups of bacteria,[3] and also some Archaea such as Sulfolobus acidocaldarius.[11]

Schematic diagram of cell wall of Gram-negative bacteria (Pseudomonas) showing the inner membrane where PTE is anchored and the periplasmic space.

Phosphotriesterase is a membrane-associated protein that is translated with a 29 amino acid-long target peptide (Tat motif),[12][10][13] which is then cleaved from the mature protein after insertion in the plasma membrane.[1] The protein is anchored to the inner membrane of the cell, facing the periplasm.[14]

The enzyme phosphotriesterase hydrolyzes organophosphate compounds by cleaving the triester linkage in the substrate.

Organophosphate compounds that serve as substrates for enzyme-catalyzed hydrolysis by PTE.

The enzyme has a very broad substrate specificity,[12] and is very efficient in catalyzing the reaction: PTE hydrolyzes paraoxon at a rate approaching the diffusion limit,[15] which indicates that the enzyme is optimally evolved for using this substrate.[13] It acts specifically on synthetic organophosphate triesters and phosphorofluoridates.[3] It does not seem to have a natural occurring substrate and may thus have optimally evolved for utilizing paraoxon and other common agricultural pesticides.[15]

The products of the reaction are diethyl phosphoric acid and p-nitrophenol.[4] The latter product is further degraded by an enzyme encoded 750bp downstream of the opd gene, and encodes a 29kDa putative hydrolase that may be involved in degrading aromatic compounds, and works in concert with PTE.[4] This enzyme is homologous to hydrolases in Pseudomonas putida, Pseudomonas azelaica, Rhodococcus sp., and P. fluorescens.[4]

Organophosphates are not toxic to bacteria, but they act as acetylcholinesterase inhibitors in animals.[16] Some species of bacteria are also able to utilize organophosphates as a nutrient and carbon source.[14]

Phosphotriesterases are considered a strong candidate biocatalyst for bioremediation purposes.[7] Its wide substrate specificity and catalytic efficiency makes it an attractive target for the potential use of microbes containing the opd gene in detoxifying soils that are toxic due to pesticide overuse.[3] Moreover, organophosphates act as acetylcholinesterase (AChE) inhibitors. The AChE neurotransmitter is a vital component of the central nervous system (CNS) in insects in animals, and the inhibition of the proper turnover of this neurochemical results in overstimulation of the CNS, which ultimately results in death of insects and mammals.[3][17] As a result, the use of organophosphate-degrading microorganisms is a potentially effective, low-cost, and environmentally friendly method of removing these toxic compounds from the environment.[3]

Bacterial species that had the ability to degrade organophosphate pesticides have been isolated from soil samples from different parts of the world. The first bacterial strain identified to be able to hydrolyze organophosphates was Flavobacterium sp. ATCC 27551, found by Sethunathan and Yoshida in 1973 from a soil sample originally from the Philippines.[5] Since then, other species were found to also have organophosphate-degrading enzymes similar to that found in Flavobacterium[6].