Automated Fitting of Transition State Force Fields for Biomolecular Simulations

The application of the Quantum Guided Molecular Mechanics (Q2MM) method to transition states of enzymatic reactions to generate a transition state force field (TSFF) with the functional form of AMBER. The differences to fitting of small-molecule TSFFs and the similarities of the approach to transfer learning are discussed. The application to the transition state of the second hydride transfer in HMGCoA Reductase from Pseudomonas mevalonii is discussed. <br><br>

Automated Fitting of Transition State Force Fields for Biomolecular Simulations

The application of the Quantum Guided Molecular Mechanics (Q2MM) method to transition states of enzymatic reactions to generate a transition state force field (TSFF) with the functional form of AMBER. The differences to fitting of small-molecule TSFFs and the similarities of the approach to transfer learning are discussed. The application to the transition state of the second hydride transfer in HMGCoA Reductase from Pseudomonas mevalonii is discussed. <br><br>

pH dependent inhibition from ammonium ions in the Pseudomonas mevalonii HMG-CoA Reductase crystallization environment

ABSTRACTHMG-CoA reductase (Pseudomonas mevalonii) utilizes mevalonate, coenzyme A (CoA) and the cofactor NAD in a complex mechanism involving two hydride transfers with cofactor exchange, accompanied by large conformational changes by a 50 residue subdomain, to generate HMG-CoA. Details about this mechanism such as the conformational changes that allow intermediate formation, cofactor exchange and product release remain unknown. The formation of the proposed intermediates has also not been observed in structural studies with natural substrates. Having been shown to be an essential enzyme for the survival of gram-positive antibiotic resistant pathogenic bacteria, studying its mechanism in detail will be beneficial in developing novel antibacterials. The enzyme has been shown to be catalytically active inside the crystal with dithio-HMG-CoA and NADH but curiously is found to be inactive in the reverse direction in the structure bound to mevalonate, CoA and NAD.To understand the factors limiting activity in the HMGR crystal with mevalonate, CoA and NAD, we studied the effect of crystallization components and pH on enzymatic activity. We observed a strong inhibition in the crystallization buffer and an increase in activity with increasing pH. We attribute this inhibitive effect to the presence of ammonium ions present in the crystal since inhibition is also observed with several other ammonium salt buffers. Additionally, the lack of inhibition was observed in the absence of ammonium. The effect of each ligand (mevalonate, CoA and NAD) on the rate of the enzymatic reaction in the crystallization environment was further investigated by measuring their Km in the crystallization buffer. The Km measurements indicate that the hydride transfer step between NAD and mevalonate is inhibited in the crystallization environment. To test this further, we solved a crystal structure of pmHMGR bound to the post-hydride transfer intermediate (mevaldehyde) and cofactor Coenzyme A. The resulting turnover with the formation of a thiohemiacetal indicated that the crystallization environment inhibited the oxidative acylation of mevalonate and the reaction intermediate mevaldyl-CoA.

Microsecond Timescale Simulations at the Transition State of PmHMGR Predict Remote Allosteric Residues

<p>Understanding the mechanisms of enzymatic catalysis requires a detailed understanding of the complex interplay of structure and dynamics of large systems that is a challenge for both experimental and computational approaches. QM/MM methods have been extensively used to study these reactions, but the difficulties arising from the hybrid treatment of the system are well documented. More importantly, the computational demands of QM/MM simulations mean that the dynamics of the reaction can only be considered on a timescale of nanoseconds even though the conformational changes needed to react the catalytically active state happen on a much slower timescale. Here we demonstrate an alternative approach that uses transition state force fields (TSFFs) derived by the quantum-guided molecular mechanics (Q2MM) method that provides a consistent treatment of the entire system at the classical molecular mechanics level and allows simulations at the microsecond timescale. Application of this approach the second hydride transfer transition state of HMG-CoA reductase from <i>Pseudomonas mevalonii </i>(<i>Pm</i>HMGR) identified three remote residues, R396 E399 and L407, (15-27 Å away from the active site) that have a remote dynamic effect on enzyme activity. The predictions were subsequently validated experimentally via site-directed mutagenesis. These results show that microsecond timescale MD simulations of transition states are possible and can predict rather than just rationalize remote allosteric residues.</p>

Microsecond Timescale Simulations at the Transition State of PmHMGR Predict Remote Allosteric Residues

<p>Understanding the mechanisms of enzymatic catalysis requires a detailed understanding of the complex interplay of structure and dynamics of large systems that is a challenge for both experimental and computational approaches. QM/MM methods have been extensively used to study these reactions, but the difficulties arising from the hybrid treatment of the system are well documented. More importantly, the computational demands of QM/MM simulations mean that the dynamics of the reaction can only be considered on a timescale of nanoseconds even though the conformational changes needed to react the catalytically active state happen on a much slower timescale. Here we demonstrate an alternative approach that uses transition state force fields (TSFFs) derived by the quantum-guided molecular mechanics (Q2MM) method that provides a consistent treatment of the entire system at the classical molecular mechanics level and allows simulations at the microsecond timescale. Application of this approach the second hydride transfer transition state of HMG-CoA reductase from <i>Pseudomonas mevalonii </i>(<i>Pm</i>HMGR) identified three remote residues, R396 E399 and L407, (15-27 Å away from the active site) that have a remote dynamic effect on enzyme activity. The predictions were subsequently validated experimentally via site-directed mutagenesis. These results show that microsecond timescale MD simulations of transition states are possible and can predict rather than just rationalize remote allosteric residues.</p>