Head of the Tunneling Group. The main activity of the group lies on the border of molecular biology and computational chemistry. We are using advanced theoretical approach to investigate properties of various enzymes, to design biologically active compounds and to help experimentalist in interpretation of their results.
Several different approaches are used to describe the role of protein compartments and residues i... more Several different approaches are used to describe the role of protein compartments and residues in catalysis and to identify key residues suitable for the modification of the activity or selectivity of the desired enzyme. In our research, we applied a combination of molecular dynamics simulations and a water tracking approach to describe the water accessible volume of Solanum tuberosum epoxide hydrolase. Using water as a molecular probe, we were able to identify small cavities linked with the active site: (i) one made up of conserved amino acids and indispensable for the proper positioning of catalytic water and (ii) two others in which modification can potentially contribute to enzyme selectivity and activity. Additionally, we identified regions suitable for de novo tunnel design that could also modify the catalytic properties of the enzyme. The identified hot-spots extend the list of the previously targeted residues used for modification of the regioselectivity of the enzyme. Finally, we have provided an example of a simple and elegant process for the detailed description of the network of cavities and tunnels, which can be used in the planning of enzyme modifications and can be easily adapted to the study of any other protein.
The cupin-type phosphoglucose isomerase (PfPGI) from the hyperthermophilic archaeon Pyrococcus fu... more The cupin-type phosphoglucose isomerase (PfPGI) from the hyperthermophilic archaeon Pyrococcus furiosus catalyzes the reversible isomerization of glucose-6-phosphate to fructose-6-phosphate. We investigated PfPGI using protein-engineering bioinformatics tools to select functionally-important residues based on correlated mutation analyses. A pair of amino acids in the periphery of PfPGI was found to be the dominant co-evolving mutation. The position of these selected residues was found to be non-obvious to conventional protein engineering methods. We designed a small smart library of variants by substituting the co-evolved pair and screened their biochemical activity, which revealed their functional relevance. Four mutants were further selected from the library for purification, measurement of their specific activity, crystal structure determination, and metal cofactor coordination analysis. Though the mutant structures and metal cofactor coordination were strikingly similar, variations in their activity correlated with their fine-tuned dynamics and solvent access regulation. Alternative, small smart libraries for enzyme optimization are suggested by our approach, which is able to identify non-obvious yet beneficial mutations.
D-amino acid oxidase (DAAO) degrades D-amino acids to produce α-ketoacids, hydrogen peroxide and ... more D-amino acid oxidase (DAAO) degrades D-amino acids to produce α-ketoacids, hydrogen peroxide and ammonia. DAAO has often been investigated and engineered for industrial and clinical applications. We combined information from literature with a detailed analysis of the structure to engineer mammalian DAAOs. The structural analysis was complemented with molecular dynamics simulations to characterize solvent accessibility and product release mechanisms. We identified non-obvious residues located on the loops on the border between the active site and the secondary binding pocket essential for pig and human DAAO substrate specificity and activity. We engineered DAAOs by mutating such critical residues and characterised the biochemical activity of the resulting variants. The results highlight the importance of the selected residues in modulating substrate specificity, product egress and enzyme activity, suggesting further steps of DAAO re-engineering towards desired clinical and industrial applications.
ABSTRACT
Motivation:The identification and tracking of molecules which enter active site cavity ... more ABSTRACT
Motivation:The identification and tracking of molecules which enter active site cavity requires screening the positions of thousands of single molecules along several thousand molecular dynamic steps. To fill the existing gap between tools searching for tunnels and pathways and advanced tools employed for accelerated water flux investigations, we have developed AQUA-DUCT.
Results: AQUA-DUCT is an easy-to-use tool that facilitates analysis of the behaviour of molecules that penetrate any selected region in a protein. It can be used for any type of molecules e.g., water, oxygen, carbon dioxide, organic solvents, ions.
Transport of ligands between buried active sites and bulk solvent is a key step in the catalytic ... more Transport of ligands between buried active sites and bulk solvent is a key step in the catalytic cycle of many enzymes. Absence of evolutionary optimized transport tunnels is an important barrier limiting the efficiency of biocatalysts prepared by computational design. Creating a structurally defined and functional ?hole? into the protein represents an engineering challenge. Here we describe the computational design and directed evolution of a de novo transport tunnel in haloalkane dehalogenase. Mutants with a blocked native tunnel and newly opened auxiliary tunnel in a distinct part of the structure showed dramatically modified properties. The mutants with blocked tunnels acquired specificity never observed with native family members, up to 32-times increased substrate inhibition and 17-times reduced catalytic rates. Opening of the auxiliary tunnel resulted in specificity and substrate inhibition similar to the native enzyme, and the most proficient haloalkane dehalogenase reported to date (kcat = 57 s-1 with 1,2-dibromoethane at 37oC and pH=8.6). Crystallographic analysis and molecular dynamics simulations confirmed successful introduction of structur-ally defined and functional transport tunnel. Our study demonstrates that whereas we can open the transport tunnels with reasonable proficiency, we cannot accurately predict the effects of such change on the catalytic properties. We propose that one way to increase efficiency of an enzyme is the direct its substrates and products into spatially distinct tunnels. The results clearly show the benefits of enzymes with de novo transport tunnels and we anticipate that this engineering strategy will facilitate creation of a wide range of useful biocatalysts.
This study concerns quantum chemical modeling of water behaviour on V-W-O systems. It was underta... more This study concerns quantum chemical modeling of water behaviour on V-W-O systems. It was undertaken in order to validate the hypothesis that the presence of W atoms on (001) surface of the crystalline vanadia-like species, facilitates low temperature water dissociation. Quantum chemical calculations were done with the use of modern electronic structure methodology, based on the density functional theory (DFT). The program package DMol of Molecular Simulations, was applied for the calculations. The calculations were performed for small clusters representing two adjacent metal sites, in pentacoordinated oxygen environment, analogous to bipiramidal clusters, introduced in description of the (001) layers of vanadium pentoxide.
... Štepánková, V., Chaloupková, R., Brezovský, J., Khabiri, M., Ettrich, R., Dam-borský, J.: How... more ... Štepánková, V., Chaloupková, R., Brezovský, J., Khabiri, M., Ettrich, R., Dam-borský, J.: How do Organic Solvents Affect Structure and Function of Haloalkane Dehalogenases? XIV. Setkánı biochemiku a molekulárnıch biologu, 20.-21. ...
ABSTRACT In this work we present simple mechanistic study on the effect of the interaction betwee... more ABSTRACT In this work we present simple mechanistic study on the effect of the interaction between early alkanes (methane, ethane and propane) with an O- species, postulated as reactive defect site on surfaces of many catalysts. The subject is focused on mechanisms of preliminary hydrogen abstraction by O- ion radical and prospective succeeding processes. The model for DFT calculations built of an alkane molecule and O- species is used for minimum energy pathway calculations along distinct reaction coordinate defined as a distance between oxygen and attacked atom. Our results show that O- and alkanes form stable preliminary complexes but only for methane the first hydrogen abstraction has meaningful energy barrier. Stability of the complex between formed hydroxyl and alkyl radical is also substantial but only for ethane and propane it determines different routes to stable intermediate products of ODH: alcohol precursors or appropriate alkenes. Formation of negatively charged precursors of corresponding alcohols is energetically or kinetically unfavourable and is of minor importance. The number and character of ODH products for propane may strongly vary depending on reaction conditions, what makes the catalysis for its oxidation highly sensitive to surface properties.
This paper concerns the relation between surface structure of crystalline vanadia-like active spe... more This paper concerns the relation between surface structure of crystalline vanadia-like active species on vanadia–tungsta catalyst and their activity in the selective reduction of NO by ammonia to nitrogen. The investigations were performed for Ti–Sn-rutile-supported isopropoxy-derived catalyst. The SCR activity and surface species structure were determined for the freshly prepared catalyst, for the catalyst previously used in NO reduction by ammonia (320 ppm NO, 335 ppm NH3 and 2.35 vol% O2) at 573 K as well as for the catalyst previously annealed at 573 K in helium stream containing 2.35 vol% O2. The crystalline islands, exposing main V2O5 surface, with some tungsten atoms substituted for V-ones, were found, with XPS and FT Raman spectroscopy, to be present at the surface of the freshly prepared catalyst. A profound evolution of the active species during the catalyst use at 573 K was observed. Dissociative water adsorption on V5+OW6+ sites is discussed as mainly responsible for the...
Several different approaches are used to describe the role of protein compartments and residues i... more Several different approaches are used to describe the role of protein compartments and residues in catalysis and to identify key residues suitable for the modification of the activity or selectivity of the desired enzyme. In our research, we applied a combination of molecular dynamics simulations and a water tracking approach to describe the water accessible volume of Solanum tuberosum epoxide hydrolase. Using water as a molecular probe, we were able to identify small cavities linked with the active site: (i) one made up of conserved amino acids and indispensable for the proper positioning of catalytic water and (ii) two others in which modification can potentially contribute to enzyme selectivity and activity. Additionally, we identified regions suitable for de novo tunnel design that could also modify the catalytic properties of the enzyme. The identified hot-spots extend the list of the previously targeted residues used for modification of the regioselectivity of the enzyme. Finally, we have provided an example of a simple and elegant process for the detailed description of the network of cavities and tunnels, which can be used in the planning of enzyme modifications and can be easily adapted to the study of any other protein.
The cupin-type phosphoglucose isomerase (PfPGI) from the hyperthermophilic archaeon Pyrococcus fu... more The cupin-type phosphoglucose isomerase (PfPGI) from the hyperthermophilic archaeon Pyrococcus furiosus catalyzes the reversible isomerization of glucose-6-phosphate to fructose-6-phosphate. We investigated PfPGI using protein-engineering bioinformatics tools to select functionally-important residues based on correlated mutation analyses. A pair of amino acids in the periphery of PfPGI was found to be the dominant co-evolving mutation. The position of these selected residues was found to be non-obvious to conventional protein engineering methods. We designed a small smart library of variants by substituting the co-evolved pair and screened their biochemical activity, which revealed their functional relevance. Four mutants were further selected from the library for purification, measurement of their specific activity, crystal structure determination, and metal cofactor coordination analysis. Though the mutant structures and metal cofactor coordination were strikingly similar, variations in their activity correlated with their fine-tuned dynamics and solvent access regulation. Alternative, small smart libraries for enzyme optimization are suggested by our approach, which is able to identify non-obvious yet beneficial mutations.
D-amino acid oxidase (DAAO) degrades D-amino acids to produce α-ketoacids, hydrogen peroxide and ... more D-amino acid oxidase (DAAO) degrades D-amino acids to produce α-ketoacids, hydrogen peroxide and ammonia. DAAO has often been investigated and engineered for industrial and clinical applications. We combined information from literature with a detailed analysis of the structure to engineer mammalian DAAOs. The structural analysis was complemented with molecular dynamics simulations to characterize solvent accessibility and product release mechanisms. We identified non-obvious residues located on the loops on the border between the active site and the secondary binding pocket essential for pig and human DAAO substrate specificity and activity. We engineered DAAOs by mutating such critical residues and characterised the biochemical activity of the resulting variants. The results highlight the importance of the selected residues in modulating substrate specificity, product egress and enzyme activity, suggesting further steps of DAAO re-engineering towards desired clinical and industrial applications.
ABSTRACT
Motivation:The identification and tracking of molecules which enter active site cavity ... more ABSTRACT
Motivation:The identification and tracking of molecules which enter active site cavity requires screening the positions of thousands of single molecules along several thousand molecular dynamic steps. To fill the existing gap between tools searching for tunnels and pathways and advanced tools employed for accelerated water flux investigations, we have developed AQUA-DUCT.
Results: AQUA-DUCT is an easy-to-use tool that facilitates analysis of the behaviour of molecules that penetrate any selected region in a protein. It can be used for any type of molecules e.g., water, oxygen, carbon dioxide, organic solvents, ions.
Transport of ligands between buried active sites and bulk solvent is a key step in the catalytic ... more Transport of ligands between buried active sites and bulk solvent is a key step in the catalytic cycle of many enzymes. Absence of evolutionary optimized transport tunnels is an important barrier limiting the efficiency of biocatalysts prepared by computational design. Creating a structurally defined and functional ?hole? into the protein represents an engineering challenge. Here we describe the computational design and directed evolution of a de novo transport tunnel in haloalkane dehalogenase. Mutants with a blocked native tunnel and newly opened auxiliary tunnel in a distinct part of the structure showed dramatically modified properties. The mutants with blocked tunnels acquired specificity never observed with native family members, up to 32-times increased substrate inhibition and 17-times reduced catalytic rates. Opening of the auxiliary tunnel resulted in specificity and substrate inhibition similar to the native enzyme, and the most proficient haloalkane dehalogenase reported to date (kcat = 57 s-1 with 1,2-dibromoethane at 37oC and pH=8.6). Crystallographic analysis and molecular dynamics simulations confirmed successful introduction of structur-ally defined and functional transport tunnel. Our study demonstrates that whereas we can open the transport tunnels with reasonable proficiency, we cannot accurately predict the effects of such change on the catalytic properties. We propose that one way to increase efficiency of an enzyme is the direct its substrates and products into spatially distinct tunnels. The results clearly show the benefits of enzymes with de novo transport tunnels and we anticipate that this engineering strategy will facilitate creation of a wide range of useful biocatalysts.
This study concerns quantum chemical modeling of water behaviour on V-W-O systems. It was underta... more This study concerns quantum chemical modeling of water behaviour on V-W-O systems. It was undertaken in order to validate the hypothesis that the presence of W atoms on (001) surface of the crystalline vanadia-like species, facilitates low temperature water dissociation. Quantum chemical calculations were done with the use of modern electronic structure methodology, based on the density functional theory (DFT). The program package DMol of Molecular Simulations, was applied for the calculations. The calculations were performed for small clusters representing two adjacent metal sites, in pentacoordinated oxygen environment, analogous to bipiramidal clusters, introduced in description of the (001) layers of vanadium pentoxide.
... Štepánková, V., Chaloupková, R., Brezovský, J., Khabiri, M., Ettrich, R., Dam-borský, J.: How... more ... Štepánková, V., Chaloupková, R., Brezovský, J., Khabiri, M., Ettrich, R., Dam-borský, J.: How do Organic Solvents Affect Structure and Function of Haloalkane Dehalogenases? XIV. Setkánı biochemiku a molekulárnıch biologu, 20.-21. ...
ABSTRACT In this work we present simple mechanistic study on the effect of the interaction betwee... more ABSTRACT In this work we present simple mechanistic study on the effect of the interaction between early alkanes (methane, ethane and propane) with an O- species, postulated as reactive defect site on surfaces of many catalysts. The subject is focused on mechanisms of preliminary hydrogen abstraction by O- ion radical and prospective succeeding processes. The model for DFT calculations built of an alkane molecule and O- species is used for minimum energy pathway calculations along distinct reaction coordinate defined as a distance between oxygen and attacked atom. Our results show that O- and alkanes form stable preliminary complexes but only for methane the first hydrogen abstraction has meaningful energy barrier. Stability of the complex between formed hydroxyl and alkyl radical is also substantial but only for ethane and propane it determines different routes to stable intermediate products of ODH: alcohol precursors or appropriate alkenes. Formation of negatively charged precursors of corresponding alcohols is energetically or kinetically unfavourable and is of minor importance. The number and character of ODH products for propane may strongly vary depending on reaction conditions, what makes the catalysis for its oxidation highly sensitive to surface properties.
This paper concerns the relation between surface structure of crystalline vanadia-like active spe... more This paper concerns the relation between surface structure of crystalline vanadia-like active species on vanadia–tungsta catalyst and their activity in the selective reduction of NO by ammonia to nitrogen. The investigations were performed for Ti–Sn-rutile-supported isopropoxy-derived catalyst. The SCR activity and surface species structure were determined for the freshly prepared catalyst, for the catalyst previously used in NO reduction by ammonia (320 ppm NO, 335 ppm NH3 and 2.35 vol% O2) at 573 K as well as for the catalyst previously annealed at 573 K in helium stream containing 2.35 vol% O2. The crystalline islands, exposing main V2O5 surface, with some tungsten atoms substituted for V-ones, were found, with XPS and FT Raman spectroscopy, to be present at the surface of the freshly prepared catalyst. A profound evolution of the active species during the catalyst use at 573 K was observed. Dissociative water adsorption on V5+OW6+ sites is discussed as mainly responsible for the...
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Papers by Artur Gora
Motivation:The identification and tracking of molecules which enter active site cavity requires screening the positions of thousands of single molecules along several thousand molecular dynamic steps. To fill the existing gap between tools searching for tunnels and pathways and advanced tools employed for accelerated water flux investigations, we have developed AQUA-DUCT.
Results: AQUA-DUCT is an easy-to-use tool that facilitates analysis of the behaviour of molecules that penetrate any selected region in a protein. It can be used for any type of molecules e.g., water, oxygen, carbon dioxide, organic solvents, ions.
Platform and Availability: Linux, Windows, macOS, OpenBSD, http://www.aquaduct.pl
Contact: a.gora@tunnelinggroup.pl, info@aquaduct.pl
Supplementary information Supplementary data are available at Bioinformatics online.
Motivation:The identification and tracking of molecules which enter active site cavity requires screening the positions of thousands of single molecules along several thousand molecular dynamic steps. To fill the existing gap between tools searching for tunnels and pathways and advanced tools employed for accelerated water flux investigations, we have developed AQUA-DUCT.
Results: AQUA-DUCT is an easy-to-use tool that facilitates analysis of the behaviour of molecules that penetrate any selected region in a protein. It can be used for any type of molecules e.g., water, oxygen, carbon dioxide, organic solvents, ions.
Platform and Availability: Linux, Windows, macOS, OpenBSD, http://www.aquaduct.pl
Contact: a.gora@tunnelinggroup.pl, info@aquaduct.pl
Supplementary information Supplementary data are available at Bioinformatics online.