Radical Stabilization Energies for Enzyme Engineering – Tackling the Substrate Scope of the Radical Enzyme QueE

Abstract

Experimental assessment of the reaction mechanisms and profiles of radical enzymes can be severely challenging due to the reactive nature of the intermediates, and sensitivity of cofactors such as iron sulfur clusters. Here we present an enzyme-directed computational methodology for the assessment of thermodynamic reaction profiles and screening for radical stabilization energies (RSEs) for the assessment of catalytic turnovers in radical enzymes. We have applied this new screening method to the radical SAM enzyme CPH4 synthase (QueE), following a detailed molecular dynamics (MD) analysis that clarifies the role of both specific enzyme residues and bound Mg2+, Ca2+ or Na+. The MD simulations provided the basis for a statistical approach to sample different conformational outcomes. RSE calculation at the M06-2X/6-31+G* level of theory provided the most computationally cost-effective assessment of enzyme-based energies, facilitated by an initial triage using semi-empirical methods. The impact of intermolecular interactions on RSE was clearly established and application to the assessment of potential alternative substrates (focusing on radical clock type rearrangements) proposes a selection of carbon-substituted analogues that would react to afford cyclopropylcarbinyl radical intermediates, as candidates for catalytic turnover by QueE.

The effect of orientated electric fields on biologically relevant iron-sulfur clusters: Tuning redox reactivity for catalysis

“…39,40 Computational methods such as molecular dynamics simulations, density functional theory (DFT), Valence Bond Theory and Poisson-Boltzmann Equation (PBE) Solvers have advanced our understanding of the role of electrostatics in enzyme catalysis, 41,42,43 providing a more complete picture of the function of enzymes such as alpha-amylase, 44 methyltransferases 45 and QueE. 46 These methods have also been used to study specific properties such as the contribution of individual amino acids to the overall electrostatic field of a protein, 47 electrostatic steering and channeling, 48,49 and the direct effect of the electrostatic field on catalytic rate. 50 Direct study of electric fields in the context of manipulating catalysis is a growing area of research.…”