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).
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.
Mendel
Sharon
S
Department of Chemistry, University of Warwick, Coventry CV4 7AL, UK.
Arndt
Andrew
A
Bugg
Timothy D H
TD
D001665:Binding Sites / Q000235:genetics; D002384:Catalysis; D050548:Catechol 1,2-Dioxygenase; D049308:Dioxygenases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006639:Histidine / Q000235:genetics*; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D007783:Lactones / Q000737:chemistry / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D010105:Oxygenases / Q000737:chemistry* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism
D029968:Escherichia coli Proteins; D007783:Lactones; D011993:Recombinant Fusion Proteins; D006639:Histidine; D010105:Oxygenases; D049308:Dioxygenases; D050548:Catechol 1,2-Dioxygenase; C083582:3-carboxyethylcatechol 2,3-dioxygenase; C439587:extradiol dioxygenase