Evolution of an organophosphate-degrading enzyme: a comparison of natural and directed evolution

Abstract

Organophosphate-degrading enzyme from Agrobacterium radiobacter P230 (OPDA) is a recently discovered enzyme that degrades a broad range of organophosphates. It is very similar to OPH first isolated from Pseudomonas diminuta MG. Despite a high level of sequence identity, OPH and OPDA exhibit different substrate specificities. We report here the structure of OPDA and identify regions of the protein that are likely to give it a preference for substrates that have shorter alkyl substituents. Directed evolution was used to evolve a series of OPH mutants that had activities similar to those of OPDA. Mutants were selected for on the basis of their ability to degrade a number of substrates. The mutations tended to cluster in particular regions of the protein and in most cases, these regions were where OPH and OPDA had significant differences in their sequences.

Spectroscopic and mechanistic studies of dinuclear metallohydrolases and their biomimetic complexes

“…2,36,[43][44][45][46][47] Some phosphoesterases such as the organophosphate-degrading agent OpdA have apparently recently evolved due to the widespread use of organophosphate pesticides. 48,49 Another well studied enzyme of this type is the PTE from P. diminuta and Flavobacterium species, which is also known under the name OPH (organophosphorus hydrolase). 50,51 It is proposed that these enzymes provide bacteria living in soil with essential nutrients like phosphate by hydrolysing pesticides.…”

Directed evolution of mammalian paraoxonases PON1 and PON3 for bacterial expression and catalytic specialization

“…4). This substrate, which is a close homologue of the insecticide coumaphos previously used for the directed evolution of a bacterial phosphotriesterase (28,29), exhibits higher fluorescence at a more convenient wavelength. Screening of Ϸ10 3 to 10 4 colonies from each library with 2NA yielded variants with mildly improved activity against OP substrates.…”

The enzymatic basis for pesticide bioremediation

“…For this reason, considerable effort has been made to improve its activity through in vitro evolution. Several mutations in these PTEs have been identifi ed that substantially improve the catalysis of a range of pesticides [59,99,100]. Contrary to expectation, these mutations seemingly had little effect on the substrate binding pocket and catalytic machinery of PTEs.…”

“…Both OPH and OpdA display extraordinary catalytic effi ciency for OPs, vastly superior to that of the E3 mutants described above; for instance, the k cat /K m of OpdA for the pesticide methyl parathion is in the order of 3 × 10 6 sec -1 M -1 [59]. The rapid turnover of OPs is made possible through the use of a binuclear metal active site, which has been characterised crystallographically [60].…”

“…Instead, mutations were almost uniformly located on the surface of the enzyme, remote from the active site, and found to result in global increases in the turnover rate. Likewise, other studies have succeeded at effectively isolating variants of PTEs with increased stability and expression [59,101].…”