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

Christian J Suess,Floriane L Martins,Christof M Jäger,Anna K Croft

doi:10.1021/acs.jcim.9b00017

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 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 S-adenosylmethione enzyme 7-carboxy-7-deazaguanine 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 semiempirical 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.

Highlights

Radical intermediates are extremely versatile for chemical functionalization and transformation reactions
C5, for example, show relative low radical stabilization energies (RSEs) values, but do not bind strongly enough or in the correct position for catalysis and are very unlikely to act as alternative substrates
Radical stabilization plays an important role in the enzymatic catalysis of radical SAM enzymes

Summary

INTRODUCTION

Radical intermediates are extremely versatile for chemical functionalization and transformation reactions. The reaction catalyzed by QueE represents a central step in queosine synthesis and facilitates the formation of the 7deazapurine scaffold through a radical-mediated ring contraction.[22] Scheme 1 shows the radical rearrangement during the catalysis of QueE propagating through an azacycloproylcarbinyl intermediate followed by NH2 elimination This mechanism has been first described as one of two potential pathways by Drennan and coworkers,[20] and was confirmed computationally by Zhu and Liu[23] and us.[21] The rearrangement of CPH4 proceeds after initial hydrogen abstraction from the C6 position through a cyclic azacyclopropylcarbinyl intermediate (3), before hydrogen reabstraction from AdoH and deamination to form the final product (6). The combination of MD sampling and RSE calculations has the potential to be used as an initial tool for in silico enzyme design of radical SAM enzymes, bringing these enzymes one step closer to efficient biotechnological applications

RESULTS AND DISCUSSION

CONCLUSION

Funding Sources