Identifikace katalytických pentád v haloalkán dehalogenáze DhmA y bakterie Mycobacterium avium N85:reakční mechanismus a molekulární evoluce.

Kód výsledku v IS VaVaI

<a href="https://www.isvavai.cz/riv?ss=detail&h=RIV%2F00216224%3A14310%2F06%3A00017450" target="_blank" >RIV/00216224:14310/06:00017450 - isvavai.cz</a>

Nalezeny alternativní kódy

RIV/00216224:14310/07:00022383

Výsledek na webu

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DOI - Digital Object Identifier

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Jazyk výsledku

angličtina

Název v původním jazyce

The Identification of Catalytic Pentad in the Haloalkane Dehalogenase DhmA from Mycobacterium avium N85: Reaction Mechanism and Molecular Evolution

Popis výsledku v původním jazyce

Haloalkane dehalogenase DhmA from Mycobacterium avium N85 showed poor expression and low stability when produced in Escherichia coli. Here we present expression DhmA in newly constructed pK4RP rhodococcal expression system in a soluble and stable form. Site-directed mutagenesis was used for the identification of a catalytic pentad, which makes up the reaction machinery of all currently known haloalkane dehalogenases. The putative catalytic triad Asp123, His279, Asp250 and the first halide-stabilizing residue Trp124 were deduced from sequence comparisons. The second stabilizing residue Trp164 was predicted from a homology model. Five point mutants in the catalytic pentad were constructed, tested for activity and were found inactive. A two-step reactionmechanism was proposed for DhmA. Evolution of different types of catalytic pentads and molecular adaptation towards the synthetic substrate 1,2-dichloroethane within the protein family is discussed.

Název v anglickém jazyce

The Identification of Catalytic Pentad in the Haloalkane Dehalogenase DhmA from Mycobacterium avium N85: Reaction Mechanism and Molecular Evolution

Popis výsledku anglicky

Haloalkane dehalogenase DhmA from Mycobacterium avium N85 showed poor expression and low stability when produced in Escherichia coli. Here we present expression DhmA in newly constructed pK4RP rhodococcal expression system in a soluble and stable form. Site-directed mutagenesis was used for the identification of a catalytic pentad, which makes up the reaction machinery of all currently known haloalkane dehalogenases. The putative catalytic triad Asp123, His279, Asp250 and the first halide-stabilizing residue Trp124 were deduced from sequence comparisons. The second stabilizing residue Trp164 was predicted from a homology model. Five point mutants in the catalytic pentad were constructed, tested for activity and were found inactive. A two-step reactionmechanism was proposed for DhmA. Evolution of different types of catalytic pentads and molecular adaptation towards the synthetic substrate 1,2-dichloroethane within the protein family is discussed.

Druh

J_x - Nezařazeno - Článek v odborném periodiku (Jimp, Jsc a Jost)

CEP obor

CE - Biochemie

OECD FORD obor

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Projekt

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Návaznosti

Z - Vyzkumny zamer (s odkazem do CEZ)

Rok uplatnění

2006

Kód důvěrnosti údajů

S - Úplné a pravdivé údaje o projektu nepodléhají ochraně podle zvláštních právních předpisů

Název periodika

JOURNAL OF STRUCTURAL BIOLOGY

ISSN

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e-ISSN

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Svazek periodika

1/2006

Číslo periodika v rámci svazku

1

Stát vydavatele periodika

US - Spojené státy americké

Počet stran výsledku

9

Strana od-do

9999

Kód UT WoS článku

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EID výsledku v databázi Scopus

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