Research On The Relationship Between Catalytic Function And Structure Of Pseudomonas Pseudoalcaligenes Methyl Parathion Hydrolase, OPHC2

Organophosphorus (OP) compounds commonly used as insecticides belong to a class of highly toxic neurotoxins. They inhibit acetylcholine esterase (AchE) in the central nervous system synapses, leading to a subsequent loss of nerve function and eventual death. It has been noticed that OPs brought about pollution for environment. Methyl parathion hydrolase (MPH, EC3.1.8.1), which specifically hydrolyzes the phosphoester bonds of OPs, reduced their toxicity. MPH belongs to the beta-lactamase protein family (PFAM accession no. PF00753) and its optimal substrate is methyl parathion. Until now, a few studies have been conducted on structure and catalytic mechanism of MPHs. Therefore, the relationship between structure and catalytic function of OPHC2was studied here for thermostability and substrate specificity of it.OPHC2, methyl parathion hydrolase, exhibits better thermostability than other MPHs. According to alignment of the sequence of OPHC2and other MPHs, we found there was no codon for cysteine in other MPHs whereas OPHC2contained two cysteines (Cys110and Cys146). The3D structural model of OPHC2carried out by homology modeling demonstrated the distances between the sulfurs of Cys110and Cys146was2.03A. It is impossible that there is a disulfide bond between these two cysteines. Furthermore, the SDS-PAGE analysis and the thiol-titration experiment all indicated that OPHC2contained a disulfide bond. In order to investigate whether the formation of disulfide bond affected the conformational stability, C110L, C110A, C146Aand C146M mutants were constructed. Acoording to the enzymatic activity analysis, the thermostabilities of mutants were not better than that of OPHC2. At both60℃and70℃, the half-life times of mutants were only50%of wild-type OPHC2. Moreover, the results of fluorescence spectra showed that the conformation of OPHC2changed upon mutation. The experimental results, combined with3D structural modeling, demonstrated that the formation of a C110-C146disulfide bond might stabilize the conformation of the OPHC2, which contributed to its thermostability.There are many kinds of organophosphorus pesticides. OPHC2can efficiently degradate some members of the family of OP compounds (such as methyl parathion, methyl paraoxon) but poorly degradate others (such as ethyl parathion, paraoxon).In this work, the emphase is to investigate what is important for substrate specificity of OPHC2and how to improve the substrate specificity of OPHC2by mutagenesis. According to the strategy of CAST (Combinatorial Active-Site Saturation Test), five mutants groups, A (M188/M191), B (L250/N251), C (E168/F171), D (E264/F265) and E (V55/L57) were designed. After screened, five mutants that showed better catalyis activity (the substrate is parathion) than OPHC2were elected. The mutant A1-5(M188L/M191S) and E6(L57V) were the best one in A library and E library, respectively. Then, a mutant AE (M188L/M191S/L57V) was constructed, which is combined mutant sites of Al-5and E6mutants. The three mutant genes were expressed in Pichia pastoris. The catalytic capacities (kcat/Km) to ethyl parathion of A1-5, E6and AE were up to2.7-fold,3.3-fold and4.2-fold of OPHC2, respectively. The analysis of structures indicated that the mutantion of residue57, residue188and residue191may provide a bigger space for the combination of parathion, which contains larger side-chain than methyl parathion.