The determination of enzyme mechanisms is a central topic in the study of biomolecular systems because they are involved in most processes in living organisms. The development of new experimental and computational biophysics methods that allow new and ever more detailed views of these processes is of fundamental importance not just from the basic science point of view, but also due to the wide range of applications of the methods and the knowledge derived from them. The goal of the proposal is to create a molecular movie where the position, movement and energy of every atom in the system followed over the course of the reaction. This will be achieved by pursuing two Specific Aims: (i) Time-Resolved Laue Crystallography of HMG-CoA Reductase (HMGR) and (ii) Simulation of the Reaction Pathway of HMGR. The application of the methodology will allow access to a level of detailed knowledge about enzyme chemistry that was not attainable up to now. The proposed approach relies on the emerging convergence of the timescales accessible by time- resolved crystallography and computational methods. We will use our recently developed photocaged cofactors to generate structural snapshots with a time resolution of 1-100 s by Laue crystallography. The ensembles of intermediate states generated by these snapshots will be deconvoluted using singular value decomposition (SVD) and connected using long timescale molecular dynamics (MD) simulations to provide structural, dynamic, and energetic insights into the complete reaction pathway. Polarizable and non- polarizable transition state force fields (TSFF) will be generated by the quantum-guided molecular mechanics (Q2MM). The use of TSFFs is 102-104 times faster than the widely used QM/MM methods, thus allowing extensive sampling, and treats the entire system at a consistent level, thus preventing the well- known problems resulting from the QM/MM interface region. Iterative cycles of crystal structure -> MD simulation -> Markov State ensemble generation -> SVD analysis of Laue data -> new time resolved structures will be used to study a complex reaction pathway, which can be broken down into smaller steps to facilitate both experimental and computational approaches. This combined methodology will be applied to the case of Pseudomonas mevalonii HMGR, which has a complex reaction mechanism involving three chemical steps, six large-scale conformational changes and two cofactor exchange steps. HMGR is of broad biomedical interest because it is the target of the widely used statins and a potential target for antibacterial treatments by new classes of antibiotics, but the methodology developed in this proposal is in principle applicable to a wide range of systems. To promote the use of the experimental and computational innovations introduced, all tool compounds and computational codes to be developed will be made available to the broader scientific community.

Public Health Relevance

The detailed study of enzyme mechanisms is a cornerstone of biophysical chemistry that, while basic in nature, has had a major impact on human health including the development of new mechanism- based drugs for a range of diseases and an understanding of the mechanism of action for existing drugs that allows the design of combination therapies. The combination of Laue crystallography and long-scale MD simulations will allow simultaneous studies of structure, dynamics and energetic studies with unprecedented detail. The application to HMG-CoA reductase, arguably the single most important drug target in western industrialized countries, will demonstrate the applicability of the methodology to an enzyme of very high mechanistic complexity. 1

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
Project #
Application #
Study Section
Special Emphasis Panel (ZRG1)
Program Officer
Wehrle, Janna P
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
University of Notre Dame
Schools of Arts and Sciences
Notre Dame
United States
Zip Code
Rosales, Anthony R; Quinn, Taylor R; Wahlers, Jessica et al. (2018) Application of Q2MM to predictions in stereoselective synthesis. Chem Commun (Camb) 54:8294-8311
Hansen, Eric; Limé, Elaine; Norrby, Per-Ola et al. (2016) Anomeric Effects in Sulfamides. J Phys Chem A 120:3677-82
Hansen, Eric; Rosales, Anthony R; Tutkowski, Brandon et al. (2016) Prediction of Stereochemistry using Q2MM. Acc Chem Res 49:996-1005