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

Abstract

Pyruvate formate-lyase (PFL) is a glycyl radical enzyme (GRE) playing a pivotal role in the metabolism of strict and facultative anaerobes. Its activation is carried out by a PFL-activating enzyme, a member of the radical S-adenosylmethionine (rSAM) superfamily of metalloenzymes, which introduces a glycyl radical into the Gly radical domain of PFL. The activation mechanism is still not fully understood and is structurally based on a complex with a short model peptide of PFL. Here, we present extensive molecular dynamics simulations in combination with quantum mechanics/molecular mechanics (QM/MM)-based kinetic and thermodynamic reaction evaluations of a more complete activation model comprising the 49 amino acid long C-terminus region of PFL. We reveal the benefits and pitfalls of the current activation model, providing evidence that the bound peptide conformation does not resemble the bound protein–protein complex conformation with PFL, with implications for the activation process. Substitution of the central glycine with (S)- and (R)-alanine showed excellent binding of (R)-alanine over unstable binding of (S)-alanine. Radical stabilization calculations indicate that a higher radical stability of the glycyl radical might not be the sole origin of the evolutionary development of GREs. QM/MM-derived radical formation kinetics further demonstrate feasible activation barriers for both peptide and C-terminus activation, demonstrating why the crystalized model peptide system is an excellent inhibitory system for natural activation. This new evidence supports the theory that GREs converged on glycyl radical formation due to the better conformational accessibility of the glycine radical loop, rather than the highest radical stability of the formed peptide radicals.

Benzylic Radical Stabilization Permits Ether Formation During Darobactin Biosynthesis

“…Previous theoretical analyses have investigated the rSAM enzyme mechanism. [69][70][71] However, our studies illustrate how substrate radical stability and transition state structure correlates with the DarE reaction outcome. These insights should allow the rational engineering of darobactin variants, and perhaps any rSAM-installed cyclophane, to contain a desired crosslink type.…”

Darobactin Substrate Engineering and Computation Show Radical Stability Governs Ether versus C–C Bond Formation

“…To better understand ether versus C–C cross-link formation, we performed extensive quantum mechanical calculations and enzyme docking studies. Previous theoretical analyses have investigated rSAM enzyme mechanisms, and our studies illustrate how substrate radical stability and transition state structure correlate with the DarE reaction outcome. − We acknowledge that the QM calculations presented are somewhat limited, as they focus exclusively on the near-attack conformation of the substrates and do not account for the role of DarE in fine-tuning the cross-link differentiation preferences of DarA variants. A comprehensive description would account for the flexibility of DarA, DarE, SAM, and the full network of solvent interactions for the entire reaction coordinate.…”

If It Is Hard, It Is Worth Doing: Engineering Radical Enzymes from Anaerobes

“…One area where this approach has been usefully exploited has been by calculating radical stabilities. Radical stabilities can be significantly modified by the encapsulating protein environment. − An initial assessment of the influence of structural variation can be made through minimal active site models. Selected ab initio and/or DFT calculations are then carried out on a subset of key residues supported by crystallographic studies, and an assessment of accessible and tractable substrates and intermediates can be made.…”