Halide-stabilizing residues of haloalkane dehalogenases studied
by quantum mechanic calculations and site-directed mutagenesis

J 2002

Halide-stabilizing residues of haloalkane dehalogenases studied by quantum mechanic calculations and site-directed mutagenesis

BOHÁČ, Michal, Yuji NAGATA, Zbyněk PROKOP, Martin PROKOP, Marta MONINCOVÁ et. al.

Basic information

Original name

Halide-stabilizing residues of haloalkane dehalogenases studied by quantum mechanic calculations and site-directed mutagenesis

Authors

BOHÁČ, Michal (203 Czech Republic), Yuji NAGATA (392 Japan), Zbyněk PROKOP (203 Czech Republic), Martin PROKOP (203 Czech Republic), Marta MONINCOVÁ (203 Czech Republic), Masataka TSUDA (392 Japan), Jaroslav KOČA (203 Czech Republic) and Jiri DAMBORSKÝ (203 Czech Republic, guarantor)

Edition

Biochemistry, 2002, 0006-2960

Other information

Language

English

Type of outcome

Článek v odborném periodiku

Field of Study

10600 1.6 Biological sciences

Country of publisher

United States of America

Confidentiality degree

není předmětem státního či obchodního tajemství

References:

URL

Impact factor

Impact factor: 4.064

RIV identification code

RIV/00216224:14310/02:00006914

Organization unit

Faculty of Science

UT WoS

000179517000022

Keywords in English

QUANTUM MECHANICS; HALIDE; MUTANT; PROTEIN ENGINEERING; STABILISATION

Abstract

V originále

Haloalkane dehalogenases catalyze cleavage of the carbon-halogen bond in halogenated aliphatic compounds resulting in the formation of an alcohol, a halide and a proton as the reaction products. Three structural features of haloalkane dehalogenases are essential for their catalytic performance: (i) a catalytic triad, (ii) an oxyanion hole and (iii) the halide-stabilizing residues. Halide-stabilizing residues are not structurally conserved among different haloalkane dehalogenases. The level of stabilization of the transition state structure of SN2 reaction and halide ion provided by each of the active site residues in the enzymes DhlA, LinB and DhaA was quantified by quantum mechanic calculations. The residues that significantly stabilize the halide ion were assigned as the primary (essential) or the secondary (less important) halide-stabilizing residues. Site-directed mutagenesis was conducted with LinB enzyme to confirm location of its primary halide-stabilizing residues. Asn38Asp, Asn38Glu, Asn38Phe, Asn38Gln, Trp109Leu, Phe151Leu, Phe151Trp, Phe151Tyr and Phe169Leu mutants of LinB were constructed, purified and kinetically characterized. The following active site residues were classified as the primary halide-stabilizing residues: Trp125 and Trp175 of DhlA; Asn38 and Trp109 of LinB; and Asn41 and Trp107 of DhaA. All these residues make a hydrogen bond with the halide ion released from the substrate molecule and their substitution results in enzymes with significantly modified catalytic properties. The following active site residues were classified as the secondary halide-stabilizing residues: Phe172, Pro223 and Val226 of DhlA; Trp207, Pro208 and Ile211 of LinB; and Phe205, Pro206 and Ile209 of DhaA. The differences in the halide stabilizing residues of three haloalkane dehalogenases are discussed in the light of molecular adaptation of these enzymes to their substrates.

Links

ME 276, research and development project

Name: Racionální re-design mikrobiálních enzymů podílejících se na degradaci toxických organických polutantů

Investor: Ministry of Education, Youth and Sports of the CR, Rational re-design of microbial enzymes involved in degradation of toxic organic pollutants.

MSM 143100005, plan (intention)

Name: Strukturně-funkční vztahy biomolekul a jejich role v metabolismu

Investor: Ministry of Education, Youth and Sports of the CR, Biomolecular Structure-function Relationships and their role in the Metabolism

Citovat

BOHÁČ, Michal, Yuji NAGATA, Zbyněk PROKOP, Martin PROKOP, Marta MONINCOVÁ, Masataka TSUDA, Jaroslav KOČA and Jiri DAMBORSKÝ. Halide-stabilizing residues of haloalkane dehalogenases studied by quantum mechanic calculations and site-directed mutagenesis. Biochemistry. 2002, vol. 41, No 48, p. 14272-14280. ISSN 0006-2960.

@article{407283,
   author = {Boháč, Michal and Nagata, Yuji and Prokop, Zbyněk and Prokop, Martin and Monincová, Marta and Tsuda, Masataka and Koča, Jaroslav and Damborský, Jiri},
   article_number = {48},
   keywords = {QUANTUM MECHANICS; HALIDE; MUTANT; PROTEIN ENGINEERING; STABILISATION},
   language = {eng},
   issn = {0006-2960},
   journal = {Biochemistry},
   title = {Halide-stabilizing residues of haloalkane dehalogenases studied by quantum mechanic calculations and site-directed mutagenesis},
   url = {http://ncbr.chemi.muni.cz/~jiri/ABSTRACTS/biochem02b.html},
   volume = {41},
   year = {2002}
}

TY  - JOUR
ID  - 407283
AU  - Boháč, Michal - Nagata, Yuji - Prokop, Zbyněk - Prokop, Martin - Monincová, Marta - Tsuda, Masataka - Koča, Jaroslav - Damborský, Jiri
PY  - 2002
TI  - Halide-stabilizing residues of haloalkane dehalogenases studied by quantum mechanic calculations and site-directed mutagenesis
JF  - Biochemistry
VL  - 41
IS  - 48
SP  - 14272
EP  - 14272
SN  - 00062960
KW  - QUANTUM MECHANICS
KW  - HALIDE
KW  - MUTANT
KW  - PROTEIN ENGINEERING
KW  - STABILISATION
UR  - http://ncbr.chemi.muni.cz/~jiri/ABSTRACTS/biochem02b.html
N2  - Haloalkane dehalogenases catalyze cleavage of the carbon-halogen bond in halogenated aliphatic compounds resulting in the formation of an alcohol, a halide and a proton as the reaction products. Three structural features of haloalkane dehalogenases are essential for their catalytic performance: (i) a catalytic triad, (ii) an oxyanion hole and (iii) the halide-stabilizing residues. Halide-stabilizing residues are not structurally conserved among different haloalkane dehalogenases. The level of stabilization of the transition state structure of SN2 reaction and halide ion provided by each of the active site residues in the enzymes DhlA, LinB and DhaA was quantified by quantum mechanic calculations. The residues that significantly stabilize the halide ion were assigned as the primary (essential) or the secondary (less important) halide-stabilizing residues. Site-directed mutagenesis was conducted with LinB enzyme to confirm location of its primary halide-stabilizing residues. Asn38Asp, Asn38Glu, Asn38Phe, Asn38Gln, Trp109Leu, Phe151Leu, Phe151Trp, Phe151Tyr and Phe169Leu mutants of LinB were constructed, purified and kinetically characterized. The following active site residues were classified as the primary halide-stabilizing residues: Trp125 and Trp175 of DhlA; Asn38 and Trp109 of LinB; and Asn41 and Trp107 of DhaA. All these residues make a hydrogen bond with the halide ion released from the substrate molecule and their substitution results in enzymes with significantly modified catalytic properties. The following active site residues were classified as the secondary halide-stabilizing residues: Phe172, Pro223 and Val226 of DhlA; Trp207, Pro208 and Ile211 of LinB; and Phe205, Pro206 and Ile209 of DhaA. The differences in the halide stabilizing residues of three haloalkane dehalogenases are discussed in the light of molecular adaptation of these enzymes to their substrates.
ER  -

BOHÁČ, Michal, Yuji NAGATA, Zbyněk PROKOP, Martin PROKOP, Marta MONINCOVÁ, Masataka TSUDA, Jaroslav KOČA and Jiri DAMBORSKÝ. Halide-stabilizing residues of haloalkane dehalogenases studied by quantum mechanic calculations and site-directed mutagenesis. \textit{Biochemistry}. 2002, vol.~41, No~48, p.~14272-14280. ISSN~0006-2960.

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