Detail
Article
Online article
FT
Medvik - BMC
  • Something wrong with this record ?

Fluorescent substrates for haloalkane dehalogenases: Novel probes for mechanistic studies and protein labeling

V. Dockalova, EM. Sanchez-Carnerero, Z. Dunajova, E. Palao, M. Slanska, T. Buryska, J. Damborsky, P. Klán, Z. Prokop,

. 2020 ; 18 (-) : 922-932. [pub] 20200408

Language English Country Netherlands

Document type Journal Article

Persistent link   https://www.medvik.cz/link/bmc20019354

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.

References provided by Crossref.org

000      
00000naa a2200000 a 4500
001      
bmc20019354
003      
CZ-PrNML
005      
20201123123543.0
007      
ta
008      
201103s2020 ne f 000 0|eng||
009      
AR
024    7_
$a 10.1016/j.csbj.2020.03.029 $2 doi
035    __
$a (PubMed)32346465
040    __
$a ABA008 $b cze $d ABA008 $e AACR2
041    0_
$a eng
044    __
$a ne
100    1_
$a Dockalova, Veronika $u Loschmidt Laboratories, Department of Experimental Biology and RECETOX, Masaryk University, Kamenice 5/A13, 625 00 Brno, Czech Republic.
245    10
$a Fluorescent substrates for haloalkane dehalogenases: Novel probes for mechanistic studies and protein labeling / $c V. Dockalova, EM. Sanchez-Carnerero, Z. Dunajova, E. Palao, M. Slanska, T. Buryska, J. Damborsky, P. Klán, Z. Prokop,
520    9_
$a 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.
655    _2
$a časopisecké články $7 D016428
700    1_
$a Sanchez-Carnerero, Esther M $u Department of Chemistry and RECETOX, Masaryk University, Kamenice 5, 625 00 Brno, Czech Republic.
700    1_
$a Dunajova, Zuzana $u Loschmidt Laboratories, Department of Experimental Biology and RECETOX, Masaryk University, Kamenice 5/A13, 625 00 Brno, Czech Republic.
700    1_
$a Palao, Eduardo $u Department of Chemistry and RECETOX, Masaryk University, Kamenice 5, 625 00 Brno, Czech Republic.
700    1_
$a Slanska, Michaela $u Loschmidt Laboratories, Department of Experimental Biology and RECETOX, Masaryk University, Kamenice 5/A13, 625 00 Brno, Czech Republic.
700    1_
$a Buryska, Tomas $u Loschmidt Laboratories, Department of Experimental Biology and RECETOX, Masaryk University, Kamenice 5/A13, 625 00 Brno, Czech Republic.
700    1_
$a Damborsky, Jiri $u Loschmidt Laboratories, Department of Experimental Biology and RECETOX, Masaryk University, Kamenice 5/A13, 625 00 Brno, Czech Republic. International Clinical Research Center, St. Anne's University Hospital, Pekarska 53, 656 91 Brno, Czech Republic.
700    1_
$a Klán, Petr $u Department of Chemistry and RECETOX, Masaryk University, Kamenice 5, 625 00 Brno, Czech Republic.
700    1_
$a Prokop, Zbynek $u Loschmidt Laboratories, Department of Experimental Biology and RECETOX, Masaryk University, Kamenice 5/A13, 625 00 Brno, Czech Republic. International Clinical Research Center, St. Anne's University Hospital, Pekarska 53, 656 91 Brno, Czech Republic.
773    0_
$w MED00184782 $t Computational and structural biotechnology journal $x 2001-0370 $g Roč. 18, č. - (2020), s. 922-932
856    41
$u https://pubmed.ncbi.nlm.nih.gov/32346465 $y Pubmed
910    __
$a ABA008 $b sig $c sign $y a $z 0
990    __
$a 20201103 $b ABA008
991    __
$a 20201123123542 $b ABA008
999    __
$a ind $b bmc $g 1586132 $s 1109552
BAS    __
$a 3
BAS    __
$a PreBMC
BMC    __
$a 2020 $b 18 $c - $d 922-932 $e 20200408 $i 2001-0370 $m Computational and structural biotechnology journal $n Comput Struct Biotechnol J $x MED00184782
LZP    __
$a Pubmed-20201103

Find record

Citation metrics

Archiving options