QM/MM Description of Newly Selected Catalytic Bioscavengers Against Organophosphorus Compounds Revealed Reactivation Stimulus Mediated by Histidine Residue in the Acyl-Binding Loop

Abstract

Butyrylcholinesterase (BChE) is considered as an efficient stoichiometric antidote against organophosphorus (OP) poisons. Recently we utilized combination of calculations and ultrahigh-throughput screening (uHTS) to select BChE variants capable of catalytic destruction of OP pesticide paraoxon. The purpose of this study was to elucidate the molecular mechanism underlying enzymatic hydrolysis of paraoxon by BChE variants using hybrid quantum mechanical/molecular mechanical (QM/MM) calculations. Detailed analysis of accomplished QM/MM runs revealed that histidine residues introduced into the acyl-binding loop are always located in close proximity with aspartate residue at position 70. Histidine residue acts as general base thus leading to attacking water molecule activation and subsequent SN2 inline hydrolysis resulting in BChE reactivation. This combination resembles canonical catalytic triad found in active centers of various proteases. Carboxyl group activates histidine residue by altering its pK_a, which in turn promotes the activation of water molecule in terms of its nucleophilicity. Observed re-protonation of catalytic serine residue at position 198 from histidine residue at position 438 recovers initial configuration of the enzyme's active center, facilitating next catalytic cycle. We therefore suggest that utilization of uHTS platform in combination with deciphering of molecular mechanisms by QM/MM calculations may significantly improve our knowledge of enzyme function, propose new strategies for enzyme design and open new horizons in generation of catalytic bioscavengers against OP poisons.

Keywords: bioscavenger; butyrylcholinesterase; computer design; organophosphorus compound; paraoxon; ultrahigh-throughput screening.

FIGURE 1

(A) The scheme of the universal ultrahigh-throughput screening (uHTS) platform for biocatalysts selection. For the selected biocatalyst (1) the gene library is constructed (2). The library is further cloned into the specialized vectors (3) and transformed into the Pichia pastoris yeast cells, that present biocatalysts on the cell surface (4). The cells with displayed biocatalysts are screened by uHTS platform: (5) single cells are compartmentalized in the aqueous droplets of a double w/o/w emulsion with the fluorogenic substrate utilizing microfluidic technology. (6) Droplets with cells expressing active enzyme and therefore containing the fluorescent product are sorted by FACS. (B) Optimization of the plasmid vector for cloning of the BChE library for expression of the enzyme on the yeast cell surface. Scheme of the genetic constructs for expression of enzyme exposed on the cell surface (top). Comparative analysis of the surface-displayed BChE expressed under control of different promoters (left panel) and amount of BChE displayed on the cell surface promoted by different polypeptide anchors (right panel). (C) Extracellular release of the RFP, fused with different signal peptides. The genetic construct is presented on top. (D) Staining of yeast cells transformed by genetic construct (shown on top) by polyclonal anti-BChE antibodies (green fluorescence). Red fluorescence corresponds to the intracellular expression of the mCherry. (E) The scheme of the optimized genetic construct coding for the BChE variants used for uHTS (top). Analysis of enzymatic activity of surface-displayed WT BChE by Ellman’s assay.

FIGURE 2

Positions of histidine residue selected after calculation and clusterization of structures corresponding to the clone 14 (A) and clone 15 (B).

FIGURE 3

Computer analysis of the catalytic mechanism of paraoxon hydrolysis by selected BChE variants. (A–E) Snapshots of the reactivation reaction path of paraoxon hydrolysis by clone 14. (F–J) Snapshots of the reactivation reaction path of paraoxon hydrolysis by clone 15.

FIGURE 4

Proposed generalized reaction mechanism of paraoxon hydrolysis by clones 14 and 15 based on trajectory snapshots. RS, reactant state; TS1, first transition state; INT, intermediate; TS2, second transition state; PS, product state.

FIGURE 5

Energy profile for clones 14 and 15. Metadynamics profile shown in solid line. Reported are energy values for reaction states with PBE0 calculations shown in parentheses.