Global and Kinetic Profiles of Substrate Diffusion in <i>Candida antarctica</i> Lipase B: Molecular Dynamics with the Markov-State Model

Lu, Chenlin; Xue, Peng; Lu, Diannan; Liu, Zheng

doi:10.1021/acsomega.9b04432

Cited by 9 publications

(6 citation statements)

References 24 publications

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“…The work also calls for further ITC studies on enzyme variants for fundamental studies to identify the involvement of positions of interest on reaction component binding or conversion . Finally, molecular modeling of the binding of reaction components can provide atomistic insights into binding processes. − Because our approach is amenable to automation and scale-up for high-throughput, the combination of such diverse approaches will provide the high-quality data needed for the engineering of enzymes and biocatalytic processes through machine learning to speed up the future development of industrial biocatalysis. ,− …”

Section: Discussionmentioning

confidence: 99%

Toward Reproducible Enzyme Modeling with Isothermal Titration Calorimetry

et al. 2021

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To apply enzymes in technical processes, a detailed understanding of the molecular mechanisms is required. Kinetic and thermodynamic parameters of enzyme catalysis are crucial to plan, model, and implement biocatalytic processes more efficiently. While the kinetic parameters, K m and k cat , are often accessible by optical methods, the determination of thermodynamic parameters requires more sophisticated methods. Isothermal titration calorimetry (ITC) allows the label-free and highly sensitive analysis of kinetic and thermodynamic parameters of individual steps in the catalytic cycle of an enzyme reaction. However, since ITC is susceptible to interferences due to denaturation or agglomeration of the enzymes, the homogeneity of the enzyme sample must always be considered, and this can be accomplished by means of dynamic light scattering (DLS) analysis. We here report on the use of an ITC-dependent work flow to determine both the kinetic and the thermodynamic data for a cofactor-dependent enzyme. Using a standardized approach with the implementation of sample quality control by DLS, we obtain high-quality data suitable for the advanced modeling of the enzyme reaction mechanism. Specifically, we investigated stereoselective reactions catalyzed by the NADPH-dependent ketoreductase Gre2p under different reaction conditions. The results revealed that this enzyme operates with an ordered sequential mechanism and is affected by substrate or product inhibition depending on the reaction buffer. Data reproducibility is ensured by specifying standard operating procedures, using programmed workflows for data analysis, and storing all data in a F.A.I.R. (findable, accessible, interoperable, and reusable) repository (https://doi.org/10.15490/fairdomhub.1.investigation.464.1). Our work highlights the utility for combined binding and kinetic studies for such complex multisubstrate reactions.

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Section: Discussionmentioning

confidence: 99%

Toward Reproducible Enzyme Modeling with Isothermal Titration Calorimetry

et al. 2021

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“…59 Finally, molecular modelling of the binding of reactants can provide atomistic insight into binding processes. [60][61][62][63] Because our approach is amenable to automation and scale-up for high-throughput, the combination of such diverse approaches will provide the high-quality data needed for the engineering of enzymes and biocatalytic processes through machine learning to speed up the future development of industrial biocatalysis. 1,[64][65][66][67]…”

Section: Discussionmentioning

confidence: 99%

Towards Reproducible Enzyme Modeling with Isothermal Titration Calorimetry

Ott¹,

Rabe²,

Niemeyer³

et al. 2021

Preprint

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<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>

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“…Another study using CMD simulation indicated that interfacial activation is due to a lamellar-like nanostructure formed by triglycerides (tricaproin and tricaprylin) in a water–triglycerides system . Furthermore, the role of the conformational dynamics of CALB during substrate binding was investigated by MD and the Markov state model (MSM) …”

Section: Introductionmentioning

confidence: 99%

“…24 Furthermore, the role of the conformational dynamics of CALB during substrate binding was investigated by MD and the Markov state model (MSM). 25 Improvements should be achieved by employing enhanced conformational sampling methods with explicit solvent models for the hydrophobic surface, followed by analysis of the freeenergy landscape concerning the lid open−close motion. In this regard, the combination of parallel cascade selection molecular dynamics (PaCS-MD), 27 an enhanced sampling method, and the Markov state model (MSM), 28−30 namely, PaCS-MD/MSM, can be used.…”

Section: ■ Introductionmentioning

confidence: 99%

Energetic and Kinetic Origins of CALB Interfacial Activation Revealed by PaCS-MD/MSM

Wijaya,

Kitao

2023

J. Phys. Chem. B

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The conformational dynamics of Candida antarctica lipase B (CALB) was investigated by molecular dynamics (MD) simulation, parallel cascade selection MD (PaCS-MD), and the Markov state model (MSM) and mainly focused on the lid-opening motion closely related to substrate binding. All-atom MD simulation of CALB was conducted in water and on the interface of water and tricaprylin. CALB initially situated in water and separated by layers of water from the interface is spontaneously adsorbed onto the tricaprylin surface during MD simulation. The opening and closing motions of the lid are simulated by PaCS-MD, and subsequent MSM analysis provided the free-energy landscape and time scale of the conformational transitions among the closed, semiopen, and open states. The closed state is the most stable in the water system, but the stable conformation in the interface system shifts to the semiopen state. These effects could explain the energetics and kinetics origin of the previously reported interfacial activation of CALB. These findings could help expand the application of CALB toward a wide variety of substrates.

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Global and Kinetic Profiles of Substrate Diffusion in Candida antarctica Lipase B: Molecular Dynamics with the Markov-State Model

Cited by 9 publications

References 24 publications

Toward Reproducible Enzyme Modeling with Isothermal Titration Calorimetry

Toward Reproducible Enzyme Modeling with Isothermal Titration Calorimetry

Towards Reproducible Enzyme Modeling with Isothermal Titration Calorimetry

Energetic and Kinetic Origins of CALB Interfacial Activation Revealed by PaCS-MD/MSM

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