EnzyHTP: A High-Throughput Computational Platform for Enzyme Modeling

Molecular simulations, including quantum mechanics (QM), molecular mechanics (MM), and multiscale QM/MM modeling, have been extensively applied to understand the mechanism of enzyme catalysis and to design new enzymes. However, molecular simulations typically require specialized, manual operation ranging from model construction to post-analysis to complete the entire life-cycle of enzyme modeling. The dependence on manual operation makes it challenging to simulate enzymes and enzyme variants in a high-throughput fashion. In this work, we developed a Python software, EnzyHTP, to automate molecular model construction, QM, MM, and QM/MM computation, and analyses of modeling data for enzyme simulations. To test the EnzyHTP, we used fluoroacetate dehalogenase (FAcD) as a model system and simulated the enzyme interior electrostatics for 100 FAcD mutants with a random single amino acid substitution. For each enzyme mutant, the workflow involves structural model construction, 1 ns molecular dynamics simulations, and quantum mechnical calculations in 100 MD-sampled snapshots. The entire simulation workflow for 100 mutants was completed in 7 hours with 10 GPUs and 160 CPUs. EnzyHTP is expected to improve the efficiency and reproducibility of computational enzyme, facilitate the fundamental understanding of catalytic origins across enzyme families, and accelerate the optimization of biocatalysts for non-native substrate transformation.

Towards Reproducible Enzyme Modeling with Isothermal Titration Calorimetry

<div> <p>An experimental workflow to provide detailed information of the molecular mechanisms of enzymes is described. This workflow will help in the application of enzymes in technical processes by providing crucial parameters needed to plan, model and implement biocatalytic processes more efficiently. These parameters are homogeneity of the enzyme sample (HES), kinetic and thermodynamic parameters of enzyme kinetics and binding of reactants to enzymes. The techniques used to measure these properties are dynamic light scattering (DLS), UV-Vis spectrophotometry and isothermal titration calorimetry (ITC) respectively. The workflow is standardized by the use of SOPs and python-scripted data analysis. </p> <p>We have used the NADPH-dependent alcohol dehydrogenase Gre2p as a challenging enzyme to demonstrate the power of this workflow. Our work highlights the utility for combined binding and kinetic studies for such complex multi-substrate reactions and the importance of sample quality control during experiments.</p> </div>

Towards Reproducible Enzyme Modeling with Isothermal Titration Calorimetry

<div> <p>An experimental workflow to provide detailed information of the molecular mechanisms of enzymes is described. This workflow will help in the application of enzymes in technical processes by providing crucial parameters needed to plan, model and implement biocatalytic processes more efficiently. These parameters are homogeneity of the enzyme sample (HES), kinetic and thermodynamic parameters of enzyme kinetics and binding of reactants to enzymes. The techniques used to measure these properties are dynamic light scattering (DLS), UV-Vis spectrophotometry and isothermal titration calorimetry (ITC) respectively. The workflow is standardized by the use of SOPs and python-scripted data analysis. </p> <p>We have used the NADPH-dependent alcohol dehydrogenase Gre2p as a challenging enzyme to demonstrate the power of this workflow. Our work highlights the utility for combined binding and kinetic studies for such complex multi-substrate reactions and the importance of sample quality control during experiments.</p> </div>

Determination of Total Folates in Complex Nutritional Drinks and Supplements Using a Tri-Enzyme Microbiological Method and Michaelis-Menten Kinetics

Background: Recent development of LC methods for the determination of total folates (vitamin B9) in complex matrixes have been hindered by vitamer interconversion and yield variability. The official microbiological method (AOAC Official Methods of Analysis 944.12 and 960.46) uses an end point turbidity reading to determine folate concentration. However, when measuring complex matrixes, shifts are observed in the growth curves of the microorganism and inaccuracies are introduced to this quantification method. Objective/Methods: In addition to the tri-enzyme digestion of the standard microbiological method, we have applied enzyme modeling of the initial velocity of bacterial growth using Michaelis-Menten kinetics to achieve more accurate and reproducible determinations of total folates. Results/Conclusions: Accuracy determined through spike recovery in Infant/Adult Nutritional Drink and a complex vitamin matrix gave values acceptable to AOAC standards of 85–110%. Repeatability of the low mass fraction analyte measured at micrograms per 100 g yielded relative standard deviations &lt;15% for all matrixes tested, including three standard reference materials.