Computationally guided modifications of CviUPO to improve catalytic activity

Technical Summary: Computationally Guided Modifications of CviUPO to Improve Catalytic Activity

Problem Statement
Unspecific peroxygenases (UPOs) are promising biocatalysts capable of selectively oxyfunctionalizing saturated hydrocarbons using only hydrogen peroxide ($H_2O_2$) as a co-substrate. However, their industrial application is hindered by peroxide-induced enzyme inactivation, a process likely triggered by hydroxyl radicals oxidizing amino acids in the active pocket. While targeted enzyme engineering is essential to mitigate this and enhance performance, the specific influence of active site amino acids on the catalytic cycle remains poorly understood. This study addresses the need to elucidate how modifications to key active center residues affect the energy barriers and catalytic efficiency of Collariella virescens UPO (CviUPO).

Methodology
The authors employed a synergistic computational workflow combining Molecular Dynamics (MD) simulations and Quantum Mechanics/Molecular Mechanics (QM/MM) calculations to investigate two specific mutations in CviUPO:

1. Glu162 $\to$ Asp162: Replacing the glutamic acid (involved in proton abstraction) with the shorter aspartic acid to test if increased pocket volume improves activity.
2. Cys19 $\to$ His19: Replacing the heme-anchoring cysteine with histidine to investigate potential resistance to heme bleaching or a shift toward peroxidase-like behavior.

The study utilized the following computational techniques:

• Classical MD Simulations: 120 ns simulations were performed on native, Asp-modified, and His-modified CviUPO to assess structural stability, active pocket volume, and hydration dynamics. Substrate (ethylbenzene/ETBE) stability was also tested via 100 ns MD simulations following docking.
• QM/MM Simulations: Using the ORCA software with the B3LYP functional (def2-TZVP basis set) and CHARMM36 force field, the study modeled the catalytic cycle up to the formation of Compound I (the reactive iron–oxo complex). This included geometry optimizations and frequency analyses for Low Spin (LS), Intermediate Spin (IS), and High Spin (HS) states.
• Nudged Elastic Band (NEB) Simulations: CI-NEB calculations were conducted to map the minimum energy paths and identify energy barriers between key intermediates (Initial State $\to$ Compound 0 $\to$ Compound I).
• Relaxed Energy Surface Scans: Performed for the native and His-modified enzymes with the substrate in the active pocket to investigate the second half of the catalytic cycle (Compound I $\to$ Compound II $\to$ Product).

Key Results

• Dynamic Behavior and Hydration: All three enzyme variants remained stable over 120 ns. While the Asp and His modifications resulted in slightly smaller active pocket volumes compared to the native enzyme, the average water molecule density within the pockets remained comparable across all variants. Notably, the His-modified enzyme showed higher temporal variation in pocket volume, suggesting potential structural instability.
• Compound I Formation (Native vs. Modified):
  • Asp Modification: The NEB simulations revealed a significantly increased energy barrier (approx. 3 eV) for the transition from the initial state to Compound 0. This is attributed to the shorter aspartic acid residue, which increases the distance the proton must travel from $H_2O_2$, hindering deprotonation. Consequently, the Asp modification is predicted to be detrimental to catalytic activity.
  • His Modification: The NEB results indicated no energy barrier between the initial state and Compound 0, suggesting facile formation of the ferric hydroperoxo complex. However, the barrier between Compound 0 and Compound I was higher (1.25–1.63 eV) than in the native enzyme. Furthermore, the final state (Compound I) was not energetically lower than Compound 0, raising the possibility that the catalytic cycle could become trapped in a local minimum.
• Spin States: For the modified enzymes, the calculated energies for LS, IS, and HS states were nearly identical, indicating that small changes in the local electronic environment significantly influence the preferred electronic configuration. In contrast, the native enzyme showed more distinct separation between spin states, with the IS state generally exhibiting the lowest energy values.
• Substrate Interaction: MD simulations confirmed that ETBE is stable in the active pockets of the native and His-modified enzymes but unstable in the Asp-modified variant.
• Reaction Pathway with Substrate: Relaxed surface scans for the His-modified enzyme showed a lower barrier for Compound II formation (0.67 eV) compared to the native enzyme (1.37 eV). However, the scan suggested a mechanistic deviation where a water molecule, rather than a direct rebound of the hydroxyl group, might facilitate product formation. This raises the possibility that the His modification alters the enzyme's mechanism, potentially converting it from a peroxygenase to a peroxidase, though this was not definitively confirmed.

Significance and Claims
The paper concludes that while computational simulations successfully elucidated the fundamental processes of the CviUPO catalytic cycle and highlighted the critical roles of active pocket hydration and spin states, they are insufficient to definitively determine whether the tested modifications increase overall catalytic activity.

• The Asp modification is computationally predicted to be unfavorable due to high energy barriers associated with proton transfer.
• The His modification presents a complex trade-off: it facilitates the initial formation of the ferric hydroperoxo complex but introduces higher barriers later in the cycle and may alter the reaction mechanism entirely.
• The authors emphasize that simulations alone cannot conclusively determine substrate specificity or confirm whether the His modification converts the enzyme into a peroxidase. They assert that a synergistic approach, connecting theoretical predictions with experimental verification, is required for efficient enzyme engineering. Preliminary experimental attempts to express the His-modified enzyme were challenging, yielding low enzyme amounts and heme loading, further underscoring the necessity of experimental validation.

The study serves as a demonstration of how high-level quantum chemical calculations, when integrated with molecular dynamics, can provide deep insights into electronic interactions and energy landscapes, guiding future rational design efforts while acknowledging the limitations of purely theoretical predictions.