Acid-base catalysis in the extradiol catechol dioxygenase reaction mechanism: site-directed mutagenesis of His-115 and His-179 in Escherichia coli 2,3-dihydroxyphenylpropionate 1,2-dioxygenase (MhpB)

Sharon Mendel, Andrew Arndt, Timothy D H Bugg

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56 Citations (Scopus)

Abstract

The extradiol catechol dioxygenases catalyze the non-heme iron(II)-dependent oxidative cleavage of catechols to 2-hydroxymuconaldehyde products. Previous studies of a biomimetic model reaction for extradiol cleavage have highlighted the importance of acid-base catalysis for this reaction. Two conserved histidine residues were identified in the active site of the class III extradiol dioxygenases, positioned within 4-5 A of the iron(II) cofactor. His-115 and His-179 in Escherichia coli 2,3-dihydroxyphenylpropionate 1,2-dioxygenase (MhpB) were replaced by glutamine, alanine, and tyrosine. Each mutant enzyme was catalytically inactive for extradiol cleavage, indicating the essential nature of these acid-base residues. Replacement of neighboring residues Asp-114 and Pro-181 gave D114N, P181A, and P181H mutant enzymes with reduced catalytic activity and altered pH/rate profiles, indicating the role of His-179 as a base and His-115 as an acid. Mutant H179Q was catalytically active for the lactone hydrolysis half-reaction, whereas mutant H115Q was inactive, implying a role for His-115 in lactone hydrolysis. A catalytic mechanism involving His-179 and His-115 as acid-base catalytic residues is proposed.

Original languageEnglish
Pages (from-to)13390-13396
Number of pages7
JournalBiochemistry
Volume43
Issue number42
Early online date2 Oct 2004
DOIs
Publication statusPublished - 26 Oct 2004

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Dioxygenases
Mutagenesis
Site-Directed Mutagenesis
Catalysis
Escherichia coli
Acids
Lactones
Hydrolysis
Iron
Catechols
Biomimetics
Enzymes
Glutamine
Histidine
Alanine
Tyrosine
Catalyst activity
Catalytic Domain
3-carboxyethylcatechol 2,3-dioxygenase
catechol

Keywords

  • Binding Sites
  • Catalysis
  • Catechol 1,2-Dioxygenase
  • Dioxygenases
  • Escherichia coli Proteins
  • Histidine
  • Hydrogen-Ion Concentration
  • Hydrolysis
  • Kinetics
  • Lactones
  • Mutagenesis, Site-Directed
  • Oxygenases
  • Recombinant Fusion Proteins
  • Journal Article
  • Research Support, Non-U.S. Gov't

Cite this

@article{45333ee974d149e2ae651a043b8fc725,
title = "Acid-base catalysis in the extradiol catechol dioxygenase reaction mechanism: site-directed mutagenesis of His-115 and His-179 in Escherichia coli 2,3-dihydroxyphenylpropionate 1,2-dioxygenase (MhpB)",
abstract = "The extradiol catechol dioxygenases catalyze the non-heme iron(II)-dependent oxidative cleavage of catechols to 2-hydroxymuconaldehyde products. Previous studies of a biomimetic model reaction for extradiol cleavage have highlighted the importance of acid-base catalysis for this reaction. Two conserved histidine residues were identified in the active site of the class III extradiol dioxygenases, positioned within 4-5 A of the iron(II) cofactor. His-115 and His-179 in Escherichia coli 2,3-dihydroxyphenylpropionate 1,2-dioxygenase (MhpB) were replaced by glutamine, alanine, and tyrosine. Each mutant enzyme was catalytically inactive for extradiol cleavage, indicating the essential nature of these acid-base residues. Replacement of neighboring residues Asp-114 and Pro-181 gave D114N, P181A, and P181H mutant enzymes with reduced catalytic activity and altered pH/rate profiles, indicating the role of His-179 as a base and His-115 as an acid. Mutant H179Q was catalytically active for the lactone hydrolysis half-reaction, whereas mutant H115Q was inactive, implying a role for His-115 in lactone hydrolysis. A catalytic mechanism involving His-179 and His-115 as acid-base catalytic residues is proposed.",
keywords = "Binding Sites, Catalysis, Catechol 1,2-Dioxygenase, Dioxygenases, Escherichia coli Proteins, Histidine, Hydrogen-Ion Concentration, Hydrolysis, Kinetics, Lactones, Mutagenesis, Site-Directed, Oxygenases, Recombinant Fusion Proteins, Journal Article, Research Support, Non-U.S. Gov't",
author = "Sharon Mendel and Andrew Arndt and Bugg, {Timothy D H}",
year = "2004",
month = "10",
day = "26",
doi = "10.1021/bi048518t",
language = "English",
volume = "43",
pages = "13390--13396",
journal = "Biochemistry",
issn = "0006-2960",
publisher = "American Chemical Society",
number = "42",

}

TY - JOUR

T1 - Acid-base catalysis in the extradiol catechol dioxygenase reaction mechanism

T2 - site-directed mutagenesis of His-115 and His-179 in Escherichia coli 2,3-dihydroxyphenylpropionate 1,2-dioxygenase (MhpB)

AU - Mendel, Sharon

AU - Arndt, Andrew

AU - Bugg, Timothy D H

PY - 2004/10/26

Y1 - 2004/10/26

N2 - The extradiol catechol dioxygenases catalyze the non-heme iron(II)-dependent oxidative cleavage of catechols to 2-hydroxymuconaldehyde products. Previous studies of a biomimetic model reaction for extradiol cleavage have highlighted the importance of acid-base catalysis for this reaction. Two conserved histidine residues were identified in the active site of the class III extradiol dioxygenases, positioned within 4-5 A of the iron(II) cofactor. His-115 and His-179 in Escherichia coli 2,3-dihydroxyphenylpropionate 1,2-dioxygenase (MhpB) were replaced by glutamine, alanine, and tyrosine. Each mutant enzyme was catalytically inactive for extradiol cleavage, indicating the essential nature of these acid-base residues. Replacement of neighboring residues Asp-114 and Pro-181 gave D114N, P181A, and P181H mutant enzymes with reduced catalytic activity and altered pH/rate profiles, indicating the role of His-179 as a base and His-115 as an acid. Mutant H179Q was catalytically active for the lactone hydrolysis half-reaction, whereas mutant H115Q was inactive, implying a role for His-115 in lactone hydrolysis. A catalytic mechanism involving His-179 and His-115 as acid-base catalytic residues is proposed.

AB - The extradiol catechol dioxygenases catalyze the non-heme iron(II)-dependent oxidative cleavage of catechols to 2-hydroxymuconaldehyde products. Previous studies of a biomimetic model reaction for extradiol cleavage have highlighted the importance of acid-base catalysis for this reaction. Two conserved histidine residues were identified in the active site of the class III extradiol dioxygenases, positioned within 4-5 A of the iron(II) cofactor. His-115 and His-179 in Escherichia coli 2,3-dihydroxyphenylpropionate 1,2-dioxygenase (MhpB) were replaced by glutamine, alanine, and tyrosine. Each mutant enzyme was catalytically inactive for extradiol cleavage, indicating the essential nature of these acid-base residues. Replacement of neighboring residues Asp-114 and Pro-181 gave D114N, P181A, and P181H mutant enzymes with reduced catalytic activity and altered pH/rate profiles, indicating the role of His-179 as a base and His-115 as an acid. Mutant H179Q was catalytically active for the lactone hydrolysis half-reaction, whereas mutant H115Q was inactive, implying a role for His-115 in lactone hydrolysis. A catalytic mechanism involving His-179 and His-115 as acid-base catalytic residues is proposed.

KW - Binding Sites

KW - Catalysis

KW - Catechol 1,2-Dioxygenase

KW - Dioxygenases

KW - Escherichia coli Proteins

KW - Histidine

KW - Hydrogen-Ion Concentration

KW - Hydrolysis

KW - Kinetics

KW - Lactones

KW - Mutagenesis, Site-Directed

KW - Oxygenases

KW - Recombinant Fusion Proteins

KW - Journal Article

KW - Research Support, Non-U.S. Gov't

U2 - 10.1021/bi048518t

DO - 10.1021/bi048518t

M3 - Article

VL - 43

SP - 13390

EP - 13396

JO - Biochemistry

JF - Biochemistry

SN - 0006-2960

IS - 42

ER -