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

Research output: Contribution to journalArticle

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

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

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