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

21 May 2019, Version 2
This content is a preprint and has not undergone peer review at the time of posting.


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.


Radical Enzymes
Radical Stabilization
Enzyme Engineering

Supplementary materials



Comments are not moderated before they are posted, but they can be removed by the site moderators if they are found to be in contravention of our Commenting Policy [opens in a new tab] - please read this policy before you post. Comments should be used for scholarly discussion of the content in question. You can find more information about how to use the commenting feature here [opens in a new tab] .
This site is protected by reCAPTCHA and the Google Privacy Policy [opens in a new tab] and Terms of Service [opens in a new tab] apply.