Radka Chaloupková

Enantis (Czechia)

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Jiřı́ Damborský

University Hospital Brno

197

Zbyněk Prokop

Masaryk University

116

Ivana Kutá Smatanová

University of South Bohemia in České Budějovice

Jan Brezovský

Masaryk University

VŠ

Veronika Štěpánková

Enantis (Czechia)

Táňa Koudeláková

RECETOX

Yuji Nagata

Tohoku University

David Bednář

Masaryk University

Michal Kutý

University of South Bohemia in České Budějovice

PŘ

Pavlína Řezáčová

Czech Academy of Sciences, Institute of Organic Chemistry and Biochemistry

Cooperative Institutions

Masaryk University

169

John Wiley & Sons (United States)

130

Hudson Institute

124

Czech Academy of Sciences

University Hospital Brno

St. Anne's University Hospital Brno

RECETOX

Central European Institute of Technology

Charles University

Czech Academy of Sciences, Institute of Organic Chemistry and Biochemistry

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Release of Halide Ions from the Buried Active Site of the Haloalkane Dehalogenase LinB Revealed by Stopped-Flow Fluorescence Analysis and Free Energy Calculations

The Journal of Physical Chemistry B (2013)

Jana Hladílková Zbyněk Prokop Radka Chaloupková Jiřı́ Damborský Pavel Jungwirth

Release of halide ions is an essential step of the catalytic cycle of haloalkane dehalogenases. Here we describe experimentally and computationally the process of release of a halide anion from the buried active site of the haloalkane dehalogenase LinB. Using stopped-flow fluorescence analysis and umbrella sampling free energy calculations, we show that the anion binding is ion-specific and follows the ordering I(-) > Br(-) > Cl(-). We also address the issue of the protonation state of the catalytic His272 residue and its effect on the process of halide release. While deprotonation of His272 increases binding of anions in the access tunnel, we show that the anionic ordering does not change with the switch of the protonation state. We also demonstrate that a sodium cation could relatively easily enter the active site, provided the His272 residue is singly protonated, and replace thus the missing proton. In contrast, Na(+) is strongly repelled from the active site containing the doubly protonated His272 residue. Our study contributes toward understanding of the reaction mechanism of haloalkane dehalogenase enzyme family. Determination of the protonation state of the catalytic histidine throughout the catalytic cycle remains a challenge for future studies.

Catalytic cycle

10.1021/jp409040u

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Stabilization of Haloalkane Dehalogenase Structure by Interfacial Interaction with Ionic Liquids

Crystals (2021)

Anastasiia Shaposhnikova Michal Kutý Radka Chaloupková Jiřı́ Damborský Ivana Kutá Smatanová

Ionic liquids attracted interest as green alternatives to replace conventional organic solvents in protein stability studies. They can play an important role in the stabilization of enzymes such as haloalkane dehalogenases that are used for biodegradation of warfare agents and halogenated environmental pollutants. Three-dimensional crystals of haloalkane dehalogenase variant DhaA80 (T148L+G171Q+A172V+C176F) from Rhodococcus rhodochrous NCIMB 13064 were grown and soaked with the solutions of 2-hydroxyethylammonium acetate and 1-butyl-3-methylimidazolium methyl sulfate. The objective was to study the structural basis of the interactions between the ionic liquids and the protein. The diffraction data were collected for the 1.25 Å resolution for 2-hydroxyethylammonium acetate and 1.75 Å resolution for 1-butyl-3-methylimidazolium methyl sulfate. The structures were used for molecular dynamics simulations to study the interactions of DhaA80 with the ionic liquids. The findings provide coherent evidence that ionic liquids strengthen both the secondary and tertiary protein structure due to extensive hydrogen bond interactions.

Rhodococcus rhodochrous

Dehalogenase

10.3390/cryst11091052

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Crystal structure of the common ancestor of haloalkane dehalogenases and Renilla luciferase (AncHLD-RLuc)

Radka Chaloupková Jenora Waterman Martin Marek Jiřı́ Damborský

Ancestor

10.2210/pdb6g75/pdb

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Engineering of haloalkane dehalogenase enantioselectivity towards βbromoalkanes: Open-solvated versus occluded-desolvated active sites

Radka Chaloupková Veronika Lišková Jan Brezovský David Bednář Zbyněk Prokop

Dehalogenase

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Structural Analysis of the Ancestral Haloalkane Dehalogenase AncLinB-DmbA

International Journal of Molecular Sciences (2021)

Andrii Mazur Pavel Grinkevich Radka Chaloupková Petra Havlíčková Barbora Kaščáková

Haloalkane dehalogenases (EC 3.8.1.5) play an important role in hydrolytic degradation of halogenated compounds, resulting in a halide ion, a proton, and an alcohol. They are used in biocatalysis, bioremediation, and biosensing of environmental pollutants and also for molecular tagging in cell biology. The method of ancestral sequence reconstruction leads to prediction of sequences of ancestral enzymes allowing their experimental characterization. Based on the sequences of modern haloalkane dehalogenases from the subfamily II, the most common ancestor of thoroughly characterized enzymes LinB from Sphingobium japonicum UT26 and DmbA from Mycobacterium bovis 5033/66 was in silico predicted, recombinantly produced and structurally characterized. The ancestral enzyme AncLinB-DmbA was crystallized using the sitting-drop vapor-diffusion method, yielding rod-like crystals that diffracted X-rays to 1.5 Å resolution. Structural comparison of AncLinB-DmbA with their closely related descendants LinB and DmbA revealed some differences in overall structure and tunnel architecture. Newly prepared AncLinB-DmbA has the highest active site cavity volume and the biggest entrance radius on the main tunnel in comparison to descendant enzymes. Ancestral sequence reconstruction is a powerful technique to study molecular evolution and design robust proteins for enzyme technologies.

10.3390/ijms222111992

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Crystal structure of putative ancestral haloalkane dehalogenase AncHLD3 (node 3)

Radka Chaloupková Jiřı́ Damborský Martin Marek

Dehalogenase

10.2210/pdb6y9f/pdb

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Recombinant ancestral enzyme ancDbjA-DbeA NOD1

Petra Bábková Radka Chaloupková Zbyněk Prokop Jiřı́ Damborský

Phylogenetically resurrected ancestor of haloalkane dehalogenase DbjA and DbeA purified to homogeneity.The results was made based on the verification of properties.

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Recombinant ancestral enzyme ancDbjA-DbeA NOD3

Petra Bábková Radka Chaloupková Zbyněk Prokop Jiřı́ Damborský

Phylogenetically resurrected ancestor of haloalkane dehalogenase DbjA and DbeA purified to homogeneity.The results was made based on the verification of properties.

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Thermostable FGF2 Polypeptide, Use thereof and Culture MediumContaining Thermostable FGF2 Polypeptide.

Jiřı́ Damborský Pavel Dvořák David Bednář Jan Brezovský Eva Šebestová

*The present invention relates to engineered Fibroblast Growth Factor 2 (FGF2, bFGF) having improved thermal stability compared to the wild-type and the use thereof in the cell biology research, regenerative medicine and related medical applications or cosmetics. The present invention further relates to a culture medium comprising FGF2 suitable for culturing a human pluripotent stem cells involving both human embryonic stem cells and induced pluripotent stem cells.

Regenerative Medicine

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How do organic solvents affecthaloalkane dehalogenases of haloalkane dehalogenases

Veronika Štěpánková Radka Chaloupková Jan Brezovský Morteza Khabiri Rüdiger Ettrich

Lecture presented by Mgr. Veronika Stěpankova on conference XIV. Setkani biochemiků a molekularnich biologů in Brno.

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