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

Abstract

Experimental assessment of catalytic 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 enzymedirected 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 CPH 4 synthase (QueE), following a detailed molecular dynamics (MD) analysis that clarifies the role of both specific enzyme residues and bound Mg 2+ , Ca 2+ 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 costeffective 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.

Activation of Glycyl Radical Enzymes─Multiscale Modeling Insights into Catalysis and Radical Control in a Pyruvate Formate-Lyase-Activating Enzyme

“…Better stabilized radicals are longer lived, more likely to “survive” the PPI-mediated activation and less likely to undergo unwanted side reactions. C α peptide backbone radicals are particularly well stabilized by spin delocalization of the planar radical center supported by the captodative effect. − Preventing the planarization of the radical center diminishes this effect as we could demonstrate recently for the radical enzyme QueE , and earlier for protected amino acids . Here, we calculated the radical stabilization energies of the different binding conformations of dipeptides and compared them with the RSE values from dipeptides in solution.…”

“…The calculation of the relative thermodynamic stability of radicals via their radical stabilization energy (RSE), as outlined in the literature for amino acids, , radicals in enzymatic catalysis in general, peptide radicals, and rSAM enzymes, , provides valuable information on the driving factors in enzymatic radical chemistry. To calculate RSEs analogous to the procedure outlined and applied extensively by Zipse and co-workers ,,− directly from molecular simulations, a combination of MD snapshots and QM calculations was applied, as shown in Figure S2 in the Supporting Information.…”

Computational Approaches: An Underutilized Tool in the Quest to Elucidate Radical SAM Dynamics

“…Molecules 2021, 26, x FOR PEER REVIEW 6 of 15 engineering alternative reactions [30]. QueE catalyzes the Mg 2+ -dependent rearrangement of 6-carboxytetrahydropterin (CPH4) to 7-carboxy-7-deazaguanine (CDG), the basic building block for more than 30 natural products in addition to the tRNA base queuosin (Figure 4A) [31].…”

“…In fact, molecular docking is often used as a preparative step for downstream MD or QM/MM analysis, and a similar evaluation of substrate scope was performed for 7-carboxy-7-deazaguanine synthase (QueE) from B. multivorans with an eye toward engineering alternative reactions [ 30 ]. QueE catalyzes the Mg 2+ -dependent rearrangement of 6-carboxytetrahydropterin (CPH 4 ) to 7-carboxy-7-deazaguanine (CDG), the basic building block for more than 30 natural products in addition to the tRNA base queuosin ( Figure 4 A) [ 31 ].…”

“…Given this requirement, docking models with appropriate positioning and radical stabilization energies (RSEs) are still insufficient to validate a given molecule as a substrate. Suess et al demonstrated that MD trajectories of seemingly suitable protein–ligand complexes in the absence of Mg 2+ showed rapid dissociation, reiterating the importance of the ion for complex stability [ 30 ]. Retention times of the natural substrate CPH 4 were also reduced upon replacement of Mg 2+ by other ions, including Ca 2+ and Na + , likely because they fail to maintain the same hydrogen bonding network.…”