Enzyme and Directed Evolution Technologies For Nerve Agent Neutralisation
Owing to the magnitude of the utilisation of organophosphorus (OPs) insecticides and the possibility of using OPs nerve agents (NA) against civilian populations, the research and development of enzymes involved in the biotransformation and detoxification of OPs has attracted considerable attention in recent years.
A number of enzymes have been identified that can catalyse the hydrolysis of OPs, including nerve agents. Two of the best characterised are Pseudomonas diminuta phosphotriesterase (PTE) and PON1, a mammalian member of the Serum paraoxonase (PONs) family. These enzymes have excellent catalytic properties towards some OPs, but relatively poor activities against others. It has been possible to alter PTE substrate specificity by rational site-mutagenesis, but with little improvements on the wild-type rates. A more successful approach has been the application of directed-evolution strategies.
The aim of the present work has been to create variants of PTE with an increased catalytic efficiency towards OPs nerve agents. To this end, a directed evolution platform was developed to enable screening for organophosphatase activity. This methodology relies on the screening of Escherichia coli colonies transformed with PTE-variant libraries.
Twelve fluorogenic NA analogues, with a 3-chloro-7-hydroxy-4-methylcoumarin leaving group, were tested for suitability as substrates for PTEs and PON1. Included in this series were analogues of the pesticides Paraoxon and Parathion, and the chemical warfare agents DFP, Dimefox, Tabun, Sarin, Cyclosarin, Soman, VX, and Russian-VX . These chemical surrogates have a similar structure but do not share the same physico-chemical properties as the nerve agents themselves.
The directed evolution platform developed and used consisted of two parts. First, partially lysed Escherichia coli colonies were screened using the fluorogenic nerve agents analogues as probes. Second, the selected (positive) clones were grown in microplates filled with liquid medium, and their organophosphatase activity was measured in vivo.
Several gene libraries were synthesised in each of which four codons of the residues forming PTE’s substrate binding site were selectively randomised. The PTE variant S5a was used as template for the libraries, as it expresses at 20-fold higher level than the wild type, in bacterial hosts, while retaining its kinetic properties for the wild-type substrate, Paraoxon. These libraries were screened using analogues of Russian-VX and Parathion as probes; approximately 106 clones were screened in total. The twenty most active variants, as determined in vivo, were expressed, purified, and their kinetic parameters for Paraoxon and the NA analogues were determined.
PTE-S5a itself hydrolysed 8/VX, 9/Sarin and 10/Russian-VX analogues between 2.5 and 3.5 times more readily than PTE-wt. In contrast, towards 11/Soman and 12/Cyclosarin analogues its activity, was only 70% of that of the wild type enzyme.
Three of the selected clones, PTE -A (I106T), C (I106L), and H (I106T/F132V/S308A/Y309W), exhibited a higher kcat than PTE-S5a towards Paraoxon. The latter exhibited a 5-fold increased in its turnover rate (31,016 s-1); this rate is higher than that of the in vitro evolved PTE-H5 (26,294 s-1).
PTE variants A (I106T), C (I106L), D (I106A/F132G), E (I106V/F132L), and F (I106L/F132lG) exhibited between 2 and 4-fold increases in their kcat/KM towards the Paraoxon analogue relative to PTE-S5a. Variants Q (G60V/I106L/ S308G), S (G60V/I106M/L303E/S308E), and T(G60V/I106S/L303P/S308G) showed between 2 and 14-fold improvements in their activities towards Russian-VX, Soman and Cyclosarin analogues. The selectivity for this latter group towards phosphonate NA analogues increased up to 107-fold, relative to the wild type PTE.
Each PTE monomer binds two divalent transition metal ions via a cluster of four histidines (His-55, His-57, His-201 and His-230) and one aspartate (Asp-301). In addition, the two metal ions are linked together by a carbamate functional group, formed by the carboxylation of the e-amino group of Lys-169 and a water (or hydroxide ion) from the solvent. A case study is presented in which using both site-directed mutagenesis and directed evolution strategies, the possibility of replacing the carboxylated lysine (Lys-169) by any other residue was assessed.