The Effect of a Unique Halide-Stabilising Residue on the
Catalytic Properties of Haloalkane Dehalogenase DatA from
Agrobacterium tumefaciens C58

J 2013

The Effect of a Unique Halide-Stabilising Residue on the Catalytic Properties of Haloalkane Dehalogenase DatA from Agrobacterium tumefaciens C58

HASAN, Khomaini; Artur Wiktor GORA; Jan BREZOVSKÝ; Radka CHALOUPKOVÁ; Hana MOSKALÍKOVÁ et. al.

Basic information

Original name

The Effect of a Unique Halide-Stabilising Residue on the Catalytic Properties of Haloalkane Dehalogenase DatA from Agrobacterium tumefaciens C58

Authors

HASAN, Khomaini (360 Indonesia, belonging to the institution); Artur Wiktor GORA (616 Poland, belonging to the institution); Jan BREZOVSKÝ (203 Czech Republic, belonging to the institution); Radka CHALOUPKOVÁ (203 Czech Republic, belonging to the institution); Hana MOSKALÍKOVÁ (203 Czech Republic, belonging to the institution); Andrea FOŘTOVÁ (203 Czech Republic, belonging to the institution); Yuji NAGATA (392 Japan); Jiří DAMBORSKÝ (203 Czech Republic, guarantor, belonging to the institution) and Zbyněk PROKOP (203 Czech Republic, belonging to the institution)

Edition

FEBS Journal, Blackwell, 2013, 1742-464X

Other information

Language

English

Type of outcome

Article in a journal

Field of Study

10600 1.6 Biological sciences

Country of publisher

United States of America

Confidentiality degree

is not subject to a state or trade secret

Impact factor

Impact factor: 3.986

RIV identification code

RIV/00216224:14310/13:00065822

Organization unit

Faculty of Science

DOI

https://doi.org/10.1111/febs.12238

UT WoS

000320557100016

Keywords in English

Haloalkane Dehalogenase

Abstract

In the original language

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.

Links

ED0001/01/01, research and development project

Name: CETOCOEN

GAP207/12/0775, research and development project

Name: Strukturně-funkční vztahy haloalkan dehalogenas

Investor: Czech Science Foundation

GAP503/12/0572, research and development project

Name: Konstrukce syntetické metabolické dráhy pro degradaci důležitého environmentálního polutantu proteinovým a metabolickým inženýrstvím

Investor: Czech Science Foundation

IAA401630901, research and development project

Name: Evoluce substrátové specifity u enzymů aktivních s xenobiotickými látkami

Investor: Academy of Sciences of the Czech Republic, Evolution of substrate specificity in enzymes acting on xenobiotic compounds

Cite

HASAN, Khomaini; Artur Wiktor GORA; Jan BREZOVSKÝ; Radka CHALOUPKOVÁ; Hana MOSKALÍKOVÁ; Andrea FOŘTOVÁ; Yuji NAGATA; Jiří DAMBORSKÝ and Zbyněk PROKOP. The Effect of a Unique Halide-Stabilising Residue on the Catalytic Properties of Haloalkane Dehalogenase DatA from Agrobacterium tumefaciens C58. FEBS Journal. Blackwell, 2013, vol. 280, No 13, p. 3149-3159. ISSN 1742-464X. Available from: https://doi.org/10.1111/febs.12238.

@article{1138865,
   author = {Hasan, Khomaini and Gora, Artur Wiktor and Brezovský, Jan and Chaloupková, Radka and Moskalíková, Hana and Fořtová, Andrea and Nagata, Yuji and Damborský, Jiří and Prokop, Zbyněk},
   article_number = {13},
   doi = {https://doi.org/10.1111/febs.12238},
   keywords = {Haloalkane Dehalogenase},
   language = {eng},
   issn = {1742-464X},
   journal = {FEBS Journal},
   title = {The Effect of a Unique Halide-Stabilising Residue on the Catalytic Properties of Haloalkane Dehalogenase DatA from Agrobacterium tumefaciens C58},
   volume = {280},
   year = {2013}
}

TY  - JOUR
ID  - 1138865
AU  - Hasan, Khomaini - Gora, Artur Wiktor - Brezovský, Jan - Chaloupková, Radka - Moskalíková, Hana - Fořtová, Andrea - Nagata, Yuji - Damborský, Jiří - Prokop, Zbyněk
PY  - 2013
TI  - The Effect of a Unique Halide-Stabilising Residue on the Catalytic Properties of Haloalkane Dehalogenase DatA from Agrobacterium tumefaciens C58
JF  - FEBS Journal
VL  - 280
IS  - 13
SP  - 3149-3159
EP  - 3149-3159
PB  - Blackwell
SN  - 1742464X
KW  - Haloalkane Dehalogenase
N2  - 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.
ER  -

HASAN, Khomaini; Artur Wiktor GORA; Jan BREZOVSKÝ; Radka CHALOUPKOVÁ; Hana MOSKALÍKOVÁ; Andrea FOŘTOVÁ; Yuji NAGATA; Jiří DAMBORSKÝ and Zbyněk PROKOP. The Effect of a Unique Halide-Stabilising Residue on the Catalytic Properties of Haloalkane Dehalogenase DatA from Agrobacterium tumefaciens C58. \textit{FEBS Journal}. Blackwell, 2013, vol.~280, No~13, p.~3149-3159. ISSN~1742-464X. Available from: https://doi.org/10.1111/febs.12238.

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