Examinando por Autor "Dockalova, Veronika"

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Fluorescent substrates for haloalkane dehalogenases: Novel probes for mechanistic studies and protein labeling
(Elsevier, 2020-04-08) Dockalova, Veronika; Sanchez-Carnerero, Esther M.; Dunajova, Zuzana; Palao, Eduardo; Slanska, Michaela; Buryska, Tomas; Damborsky, Jiri; Klán, Petr; Prokop, Zbynek
Haloalkane dehalogenases are enzymes that catalyze the cleavage of carbon-halogen bonds in halogenated compounds. They serve as model enzymes for studying structure–function relationships of >100.000 members of the α/β-hydrolase superfamily. Detailed kinetic analysis of their reaction is crucial for understanding the reaction mechanism and developing novel concepts in protein engineering. Fluorescent substrates, which change their fluorescence properties during a catalytic cycle, may serve as attractive molecular probes for studying the mechanism of enzyme catalysis. In this work, we present the development of the first fluorescent substrates for this enzyme family based on coumarin and BODIPY chromophores. Steady-state and pre-steady-state kinetics with two of the most active haloalkane dehalogenases, DmmA and LinB, revealed that both fluorescent substrates provided specificity constant two orders of magnitude higher (0.14–12.6 μM−1 s−1) than previously reported representative substrates for the haloalkane dehalogenase family (0.00005–0.014 μM−1 s−1). Stopped-flow fluorescence/FRET analysis enabled for the first time monitoring of all individual reaction steps within a single experiment: (i) substrate binding, (ii–iii) two subsequent chemical steps and (iv) product release. The newly introduced fluorescent molecules are potent probes for fast steady-state kinetic profiling. In combination with rapid mixing techniques, they provide highly valuable information about individual kinetic steps and mechanism of haloalkane dehalogenases. Additionally, these molecules offer high specificity and efficiency for protein labeling and can serve as probes for studying protein hydration and dynamics as well as potential markers for cell imaging.
The impact of tunnel mutations on enzymatic catalysis depends on the tunnel-substrate complementarity and the rate-limiting step
(Elsevier, 2020-03-25) Kokkonen, Piia; Slanska, Michaela; Dockalova, Veronika; Pinto, Gaspar P.; Sánchez-Carnerero, Esther M.; Damborsky, Jiri; Klán, Petr; Prokop, Zbynek; Bednar, David
Transport of ligands between bulk solvent and the buried active sites is a critical event in the catalytic cycle of many enzymes. The rational design of transport pathways is far from trivial due to the lack of knowledge about the effect of mutations on ligand transport. The main and an auxiliary tunnel of haloalkane dehalogenase LinB have been previously engineered for improved dehalogenation of 1,2-dibromoethane (DBE). The first chemical step of DBE conversion was enhanced by L177W mutation in the main tunnel, but the rate-limiting product release was slowed down because the mutation blocked the main access tunnel and hindered protein dynamics. Three additional mutations W140A + F143L + I211L opened-up the auxiliary tunnel and enhanced the product release, making this four-point variant the most efficient catalyst with DBE. Here we study the impact of these mutations on the catalysis of bulky aromatic substrates, 4-(bromomethyl)-6,7-dimethoxycoumarin (COU) and 8-chloromethyl-4,4′-difluoro-3,5-dimethyl-4-bora-3a,4a-diaza-s-indacene (BDP). The rate-limiting step of DBE conversion is the product release, whereas the catalysis of COU and BDP is limited by the chemical step. The catalysis of COU is mainly impaired by the mutation L177W, whereas the conversion of BDP is affected primarily by the mutations W140A + F143L + I211L. The combined computational and kinetic analyses explain the differences in activities between the enzyme-substrate pairs. The effect of tunnel mutations on catalysis depends on the rate-limiting step, the complementarity of the tunnels with the substrates and is clearly specific for each enzyme-substrate pair.