title: Enhancing the peroxidatic activity of KatG by deletion mutagenesis.
abstract: Catalase-peroxidase (KatG) enzymes use a peroxidase active site to facilitate robust catalase activity, an ability all other members of its superfamily lack. KatG's have a Met-Tyr-Trp covalent adduct that is essential for catalatic but not peroxidatic turnover. The tyrosine (Y226 in E. coli KatG) is supplied by a large loop (LL1) that is absent from all other plant peroxidases. Elimination of Y226 from the KatG structure, either by site directed mutagenesis (i.e., Y226F KatG) or by deletion of larger portions of LL1 invariably eliminates catalase activity, but deletion variants were substantially more active as peroxidases, up to an order of magnitude. Moreover, the deletion variants were more resistant to H(2)O(2)-dependent inactivation than Y226F KatG. Stopped-flow evaluation of reactions of H(2)O(2) with Y226F KatG and the most peroxidase active deletion variant (KatG[Δ209-228]) produced highly similar rate constants for formation of compounds I and II, and about a four-fold faster formation of compound III for the deletion variant as opposed to Y226F. Conversely, single turnover experiments showed a 60-fold slower return of Y226F KatG to its ferric state in the presence of the exogenous electron donor 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) than was determined for KatG(Δ209-228). Our data suggest that the peroxidatic output of KatG cannot be optimized simply by elimination of catalase activity alone, but also requires modifications that increase electron transfer between exogenous electron donors and the heme prosthetic group.
authors.affiliation: Department of Chemistry and Biochemistry, Auburn University, Auburn, AL 36849-5312, United States.
authors.forename: Shalley N
authors.initials: SN
authors.lastname: Kudalkar
authors.forename: Robert A
authors.initials: RA
authors.lastname: Campbell
authors.forename: Yongjiang
authors.initials: Y
authors.lastname: Li
authors.forename: Cornelius L
authors.initials: CL
authors.lastname: Varnado
authors.forename: Corey
authors.initials: C
authors.lastname: Prescott
authors.forename: Douglas C
authors.initials: DC
authors.lastname: Goodwin
mesh_terms: D001483:Base Sequence; D002374:Catalase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017931:DNA Primers; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D016296:Mutagenesis; D010545:Peroxides / Q000378:metabolism*; D013056:Spectrophotometry, Ultraviolet
chemical_list: D017931:DNA Primers; D029968:Escherichia coli Proteins; D010545:Peroxides; D002374:Catalase; C051253:katG protein, E coli