Similar Active Sites and Mechanisms Do Not Lead to Cross-Promiscuity in Organophosphate Hydrolysis: Implications for Biotherapeutic Engineering

Abstract

Organophosphate hydrolases are proficient catalysts of the breakdown of neurotoxic organophosphates and have great potential as both biotherapeutics for treating acute organophosphate toxicity and as bioremediation agents. However, proficient organophosphatases such as serum paraoxonase 1 (PON1) and the organophosphate-hydrolyzing lactonase SsoPox are unable to hydrolyze bulkyorganophosphates with challenging leaving groups such as diisopropyl fluorophosphate (DFP) or venomous agent X, creating a major challenge for enzyme design. Curiously, despite their mutually exclusive substrate specificities, PON1 and diisopropyl fluorophosphatase (DFPase) have essentially identical active sites and tertiary structures. In the present work, we use empirical valence bond simulations to probe the catalytic mechanism of DFPase as well as temperature, pH, and mutational effects, demonstrating that DFPase and PON1 also likely utilize identical catalytic mechanisms to hydrolyze their respective substrates. However, detailed examination of both static structures and dynamical simulations demonstrates subtle but significant differences in the electrostatic properties and solvent penetration of the two active sites and, most critically, the role of residues that make no direct contact with either substrate in acting as “specificity switches” between the two enzymes. Specifically, we demonstrate that key residues that are structurally and functionally critical for the paraoxonase activity of PON1 prevent it from being able to hydrolyze DFP with its fluoride leaving group. These insights expand our understanding of the drivers of the evolution of divergent substrate specificity in enzymes with identical active sites and guide the future design of organophosphate hydrolases that hydrolyze compounds with challenging leaving groups.

Challenges and advances in the computational modeling of biological phosphate hydrolysis

“…That is, while both DFPase and PON1 are efficient catalysts of the hydrolysis of DFP and paraoxon, respectively, they are poor catalysts of the hydrolysis of each other's substrates. 121 We performed a detailed analysis of the (lack of) cross-promiscuity between these two systems, illustrating in particular that in addition to expected structural and dynamical differences between the two enzymes, a key functional residue in PON1, D183, discussed above, is in fact severely detrimental to the DFPase activity of this enzyme due to electrostatic repulsion between the aspartate side chain and the fluoride leaving group. 121 Understanding such differences in specificity at the fine molecular detail is an important first step towards re-engineering organophosphate hydrolases that can more efficiently break down highly neurotoxic organophosphate nerve agents with challenging leaving groups such as sarin (fluoride) or VX (thiol).…”

“…Our interest in this topic, as in the case of the members of the alkaline phosphatase superfamily, has been in both mechanisms as well as in the molecular drivers for the evolution of organophosphatase activity. In this section, we will discuss some of our work in this area, 84,94,95,121 as well as the associated methodological challenges, and how were they overcome.…”

“…84 Other systems of interest to us have been serum paraoxonase 1 (PON1) 94,95 and diisopropyl fluorophosphatase (DFPase). 121 Both enzymes are six-blade b-propellers, with the active site located in the central tunnel of the propeller (Fig. 8).…”

“…94 Finally, we have performed a detailed mechanistic study of the hydrolysis of DFP by DFPase, considering not just EVB simulations of the chemical step of catalysis, but also mutational, temperature, and pH effects on the calculated activation free energies. 121 Having established that DFPase and PON1 hydrolyze organophosphates through identical chemical mechanisms, this raises the question of why two enzymes with the same mechanism and with very similar active sites are not cross-promiscuous? That is, while both DFPase and PON1 are efficient catalysts of the hydrolysis of DFP and paraoxon, respectively, they are poor catalysts of the hydrolysis of each other's substrates.…”

Rational Integration of Biomineralization, Microbial Surface Display, and Carbon Nanocomposites: Ultrasensitive and Selective Biosensor for Traces of Pesticides

“…To overcome these above problems, organophosphate hydrolase (OPH) is introduced into electrochemical OPP biosensors to substitute enzyme inhibition‐based biosensors . OPH can specifically hydrolyze organophosphate compounds, where OPP are the substrate of the enzyme rather than the inhibitor . Hence, OPH‐based analytical methods generally show good selectivity and simple assay procedure.…”