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.

Základní údaje

Originální název

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

Autoři

BOHÁČ, Michal (203 Česká republika); Yuji NAGATA (392 Japonsko); Zbyněk PROKOP (203 Česká republika); Martin PROKOP (203 Česká republika); Marta MONINCOVÁ (203 Česká republika); Masataka TSUDA (392 Japonsko); Jaroslav KOČA (203 Česká republika) a Jiri DAMBORSKÝ (203 Česká republika, garant)

Vydání

Biochemistry, 2002, 0006-2960

Další údaje

Jazyk

angličtina

Typ výsledku

Článek v odborném periodiku

Obor

10600 1.6 Biological sciences

Stát vydavatele

Spojené státy

Utajení

není předmětem státního či obchodního tajemství

Odkazy

URL

Impakt faktor

Impact factor: 4.064

Kód RIV

RIV/00216224:14310/02:00006914

Organizační jednotka

Přírodovědecká fakulta

UT WoS

000179517000022

Klíčová slova anglicky

QUANTUM MECHANICS; HALIDE; MUTANT; PROTEIN ENGINEERING; STABILISATION

Štítky

HALIDE, MUTANT, Protein engineering, quantum mechanics, STABILISATION

Změněno: 19. 3. 2010 10:54, prof. Mgr. Jiří Damborský, Dr.

Anotace

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.

Návaznosti

ME 276, projekt VaV

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

Investor: Ministerstvo školství, mládeže a tělovýchovy ČR, Racionální re-design mikrobiálních enzymů podílejících se na degradaci toxických organických polutantů

MSM 143100005, záměr

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

Investor: Ministerstvo školství, mládeže a tělovýchovy ČR, Strukturně-funkční vztahy biomolekul a jejich role v metabolismu

Citovat

BOHÁČ, Michal; Yuji NAGATA; Zbyněk PROKOP; Martin PROKOP; Marta MONINCOVÁ; Masataka TSUDA; Jaroslav KOČA a Jiri DAMBORSKÝ. Halide-stabilizing residues of haloalkane dehalogenases studied by quantum mechanic calculations and site-directed mutagenesis. Biochemistry. 2002, roč. 41, č. 48, s. 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 a Jiri DAMBORSKÝ. Halide-stabilizing residues of haloalkane dehalogenases studied by quantum mechanic calculations and site-directed mutagenesis. \textit{Biochemistry}. 2002, roč.~41, č.~48, s.~14272-14280. ISSN~0006-2960.

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