Jan Brezovsky

Publications (14) View all

• Article: Gates of Enzymes.

  Artur Gora, Jan Brezovsky, Jiri Damborsky
  Chemical Reviews 04/2013; · 40.20 Impact Factor
• Article: Expansion of Access Tunnels and Active-Site Cavities Influence Activity of Haloalkane Dehalogenases in Organic Cosolvents.

  Veronika Stepankova, Morteza Khabiri, Jan Brezovsky, Antonin Pavelka, Jan Sykora, Mariana Amaro, Babak Minofar, Zbynek Prokop, Martin Hof, Rudiger Ettrich, Radka Chaloupkova, Jiri Damborsky
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  ABSTRACT: The use of enzymes for biocatalysis can be significantly enhanced by using organic cosolvents in the reaction mixtures. Selection of the cosolvent type and concentration range for an enzymatic reaction is challenging and requires extensive empirical testing. An understanding of protein-solvent interaction could provide a theoretical framework for rationalising the selection process. Here, the behaviour of three model enzymes (haloalkane dehalogenases) was investigated in the presence of three representative organic cosolvents (acetone, formamide, and isopropanol). Steady-state kinetics assays, molecular dynamics simulations, and time-resolved fluorescence spectroscopy were used to elucidate the molecular mechanisms of enzyme-solvent interactions. Cosolvent molecules entered the enzymes' access tunnels and active sites, enlarged their volumes with no change in overall protein structure, but surprisingly did not act as competitive inhibitors. At low concentrations, the cosolvents either enhanced catalysis by lowering K0.5 and increasing kcat , or caused enzyme inactivation by promoting substrate inhibition and decreasing kcat . The induced activation and inhibition of the enzymes correlated with expansion of the active-site pockets and their occupancy by cosolvent molecules. The study demonstrates that quantitative analysis of the proportions of the access tunnels and active-sites occupied by organic solvent molecules provides the valuable information for rational selection of appropriate protein-solvent pair and effective cosolvent concentration.
  ChemBioChem 04/2013; · 3.94 Impact Factor
• Article: The Effect of a Unique Halide-Stabilising Residue on the Catalytic Properties of Haloalkane Dehalogenase DatA from Agrobacterium tumefaciens C58.

  Khomaini Hasan, Artur Gora, Jan Brezovsky, Radka Chaloupkova, Hana Moskalikova, Andrea Fortova, Yuji Nagata, Jiri Damborsky, Zbynek Prokop
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  ABSTRACT: Haloalkane dehalogenases catalyse the hydrolysis of carbon-halogen bonds in various chlorinated, brominated and iodinated compounds. These enzymes have a conserved pair of halide-stabilising residues that are important in substrate binding and stabilisation of the transition state and the halide ion product via hydrogen bonding. In all previously known haloalkane dehalogenase, these residues are either a pair of tryptophans or a tryptophan-asparagine pair. The newly isolated haloalkane dehalogenase DatA from Agrobacterium tumefaciens C58 possesses a unique halide-stabilising tyrosine residue, Y109, in place of the conventional tryptophan. A variant of DatA with the Y109W mutation was created and the effects of this mutation on the enzyme's structure and catalytic properties were studied using spectroscopy and pre-steady-state kinetic experiments. Quantum mechanical and molecular dynamics calculations were used to obtain a detailed analysis of the hydrogen bonding patterns within the active sites of the wild-type and the mutant, and of the stabilisation of the ligands as the reaction proceeds. Fluorescence quenching experiments suggested that replacing the tyrosine with tryptophan improves halide binding 3.7-fold, presumably due to the introduction of an additional hydrogen bond. Kinetic analysis revealed that the mutation affected the enzyme's substrate specificity and reduced its K0.5 for selected halogenated substrates by a factor of 2-4, without impacting the rate-determining hydrolytic step. We conclude that DatA is the first natural haloalkane dehalogenase that stabilises its substrate in the active site using only a single hydrogen bond, which is a new paradigm in catalysis by this enzyme family. © 2013 The Authors Journal compilation © 2013 FEBS.
  FEBS Journal 03/2013; · 3.79 Impact Factor
• Article: Engineering Enzyme Stability and Resistance to an Organic Cosolvent by Modification of Residues in the Access Tunnel.

  Tana Koudelakova, Radka Chaloupkova, Jan Brezovsky, Zbynek Prokop, Eva Sebestova, Martin Hesseler, Morteza Khabiri, Maryia Plevaka, Daryna Kulik, Ivana Kuta Smatanova, Pavlina Rezacova, Rudiger Ettrich, Uwe T Bornscheuer, Jiri Damborsky
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  ABSTRACT: Mutations targeting as few as four residues lining the access tunnel extended the half-life of an enzyme in 40 % dimethyl sulfoxide from minutes to weeks and increased its melting temperature by 190 °C. Protein crystallography and molecular dynamics revealed that the tunnel residue packing is a key determinant of protein stability and the active-site accessibility for cosolvent molecules (red dots).
  Angewandte Chemie International Edition 01/2013; · 13.45 Impact Factor
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  Available from: Artur Gora

  Article: Software tools for identification, visualization and analysis of protein tunnels and channels.

  Jan Brezovsky, Eva Chovancova, Artur Gora, Antonin Pavelka, Lada Biedermannova, Jiri Damborsky
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  ABSTRACT: Protein structures contain highly complex systems of voids, making up specific features such as surface clefts or grooves, pockets, protrusions, cavities, pores or channels, and tunnels. Many of them are essential for the migration of solvents, ions and small molecules through proteins, and their binding to the functional sites. Analysis of these structural features is very important for understanding of structure-function relationships, for the design of potential inhibitors or proteins with improved functional properties. Here we critically review existing software tools specialized in rapid identification, visualization, analysis and design of protein tunnels and channels. The strengths and weaknesses of individual tools are reported together with examples of their applications for the analysis and engineering of various biological systems. This review can assist users with selecting a proper software tool for study of their biological problem as well as highlighting possible avenues for further development of existing tools. Development of novel descriptors representing not only geometry, but also electrostatics, hydrophobicity or dynamics, is needed for reliable identification of biologically relevant tunnels and channels.
  Biotechnology advances 01/2013; 31:38-49. · 8.25 Impact Factor