Substrate inhibition by the blockage of product release and its control by tunnel engineering

Kokkonen, Piia; Beier, Andy; Mazurenko, Stanislav; Damborský, Jiřı́; Bednář, David; Prokop, Zbyněk

doi:10.1039/d0cb00171f

Cited by 50 publications

(35 citation statements)

References 53 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Previous studies indicated that the characteristics of substrate access tunnels can have a decisive influence on enzyme–substrate specificity and activity. − In t-PlaF, we focused on T1 and T2 because only these two allow direct access of GPL or LGPL substrates from the membrane in the t-PlaF A configuration. In contrast, to enter into T3, substrates would need to pass through the solvent, which is energetically unfavorable.…”

Section: Discussionmentioning

confidence: 99%

Substrate Access Mechanism in a Novel Membrane-Bound Phospholipase A of Pseudomonas aeruginosa Concordant with Specificity and Regioselectivity

Ahmad

Strunk

Schott‐Verdugo

et al. 2021

J. Chem. Inf. Model.

View full text Add to dashboard Cite

PlaF is a cytoplasmic membrane-bound phospholipase A 1 from Pseudomonas aeruginosa that alters the membrane glycerophospholipid (GPL) composition and fosters the virulence of this human pathogen. PlaF activity is regulated by a dimer-to-monomer transition followed by tilting of the monomer in the membrane. However, how substrates reach the active site and how the characteristics of the active site tunnels determine the activity, specificity, and regioselectivity of PlaF for natural GPL substrates have remained elusive. Here, we combined unbiased and biased all-atom molecular dynamics (MD) simulations and configurational free-energy computations to identify access pathways of GPL substrates to the catalytic center of PlaF. Our results map out a distinct tunnel through which substrates access the catalytic center. PlaF variants with bulky tryptophan residues in this tunnel revealed decreased catalysis rates due to tunnel blockage. The MD simulations suggest that GPLs preferably enter the active site with the sn-1 acyl chain first, which agrees with the experimentally demonstrated PLA 1 activity of PlaF. We propose that the acyl chain-length specificity of PlaF is determined by the structural features of the access tunnel, which results in favorable free energy of binding of medium-chain GPLs. The suggested egress route conveys fatty acid (FA) products to the dimerization interface and, thus, contributes to understanding the product feedback regulation of PlaF by FA-triggered dimerization. These findings open up opportunities for developing potential PlaF inhibitors, which may act as antibiotics against P. aeruginosa.

show abstract

Section: Discussionmentioning

confidence: 99%

Substrate Access Mechanism in a Novel Membrane-Bound Phospholipase A of Pseudomonas aeruginosa Concordant with Specificity and Regioselectivity

Ahmad

Strunk

Schott‐Verdugo

et al. 2021

J. Chem. Inf. Model.

View full text Add to dashboard Cite

show abstract

“…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

View full text Add to dashboard Cite

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.

show abstract

“…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

View full text Add to dashboard Cite

<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>

show abstract

Substrate inhibition by the blockage of product release and its control by tunnel engineering

Abstract: Substrate inhibition can be caused by substrate binding to the enzyme–product complex and can be controlled rationally by targeting enzyme access tunnels.

Cited by 50 publications

References 53 publications

Substrate Access Mechanism in a Novel Membrane-Bound Phospholipase A of Pseudomonas aeruginosa Concordant with Specificity and Regioselectivity

Substrate Access Mechanism in a Novel Membrane-Bound Phospholipase A of Pseudomonas aeruginosa Concordant with Specificity and Regioselectivity

Toward Reproducible Enzyme Modeling with Isothermal Titration Calorimetry

Towards Reproducible Enzyme Modeling with Isothermal Titration Calorimetry

Contact Info

Product

Resources

About