title: Vital roles of an interhelical insertion in catalase-peroxidase bifunctionality.
abstract: The loop connecting the F and G helices of catalase-peroxidases contains a approximately 35 amino acid structure (the FG insertion) that is absent from monofunctional peroxidases. These two groups of enzymes share highly similar active sites, yet the monofunctional peroxidases lack appreciable catalase activity. Thus, the FG insertion may serve a role in catalase-peroxidase bifunctionality, despite its peripheral location relative to the active site. We produced a variant of Escherichia coli catalase-peroxidase (KatG) lacking its FG insertion (KatG(DeltaFG)). Absorption spectra indicated the heme environment of KatG(DeltaFG) was highly similar to wild-type KatG, but the variant retained only 0.2% catalase activity. In contrast, the deletion reduced peroxidase activity by only 50%. Kinetic parameters for the peroxidase and residual catalase activities of KatG(DeltaFG) as well as pH dependence studies suggested that the FG insertion supports hydrogen-bonded networks critical for reactions involving H2O2. The structure also appears to regulate access of electron donors to the active site.
authors.affiliation: Department of Chemistry and Program in Cell and Molecular Biosciences, Auburn University, Auburn, AL 36849-5312, USA.
authors.forename: Yongjiang
authors.initials: Y
authors.lastname: Li
authors.forename: Douglas C
authors.initials: DC
authors.lastname: Goodwin
mesh_terms: D052160:Benzothiazoles; D001665:Binding Sites; D002374:Catalase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002384:Catalysis; D004926:Escherichia coli / Q000201:enzymology; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D005290:Ferric Compounds / Q000378:metabolism; D005296:Ferrous Compounds / Q000378:metabolism; D017353:Gene Deletion; D006860:Hydrogen Bonding; D006861:Hydrogen Peroxide / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009195:Peroxidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet; D013451:Sulfonic Acids / Q000378:metabolism
chemical_list: D052160:Benzothiazoles; D029968:Escherichia coli Proteins; D005290:Ferric Compounds; D005296:Ferrous Compounds; D011994:Recombinant Proteins; D013451:Sulfonic Acids; C002502:2,2'-azino-di-(3-ethylbenzothiazoline)-6-sulfonic acid; D006861:Hydrogen Peroxide; D002374:Catalase; D009195:Peroxidase