title: Manganese oxidation site in Pleurotus eryngii versatile peroxidase: a site-directed mutagenesis, kinetic, and crystallographic study.
abstract: The molecular architecture of versatile peroxidase (VP) includes an exposed tryptophan responsible for aromatic substrate oxidation and a putative Mn2+ oxidation site. The crystal structures (solved up to 1.3 A) of wild-type and recombinant Pleurotus eryngii VP, before and after exposure to Mn2+, showed a variable orientation of the Glu36 and Glu40 side chains that, together with Asp175, contribute to Mn2+ coordination. To evaluate the involvement of these residues, site-directed mutagenesis was performed. The E36A, E40A, and D175A mutations caused a 60-85-fold decrease in Mn2+ affinity and a decrease in the Mn2+ oxidation activity. Transient-state kinetic constants showed that reduction of both compounds I and II was affected (80-325-fold lower k2app and 103-104-fold lower k3app, respectively). The single mutants retained partial Mn2+ oxidation activity, and a triple mutation (E36A/E40A/D175A) was required to completely suppress the activity (<1% kcat). The affinity for Mn2+ also decreased ( approximately 25-fold) with the shorter carboxylate side chain in the E36D and E40D variants, which nevertheless retained 30-50% of the maximal activity, whereas similar mutations caused a 50-100-fold decrease in kcat in the case of the Phanerochaete chrysosporium manganese peroxidase (MnP). Additional mutations showed that introduction of a basic residue near Asp175 did not improve Mn2+ oxidation as found for MnP and ruled out an involvement of the C-terminal tail of the protein in low-efficiency oxidation of Mn2+. The structural and kinetic data obtained highlighted significant differences in the Mn2+ oxidation site of the new versatile enzyme compared to P. chrysosporium MnP.
authors.affiliation: Centro de Investigaciones Biológicas, CSIC, Ramiro de Maeztu 9, E-28040 Madrid, Spain.
authors.forename: Francisco J
authors.initials: FJ
authors.lastname: Ruiz-Dueñas
authors.forename: María
authors.initials: M
authors.lastname: Morales
authors.forename: Marta
authors.initials: M
authors.lastname: Pérez-Boada
authors.forename: Thomas
authors.initials: T
authors.lastname: Choinowski
authors.forename: María Jesús
authors.initials: MJ
authors.lastname: Martínez
authors.forename: Klaus
authors.initials: K
authors.lastname: Piontek
authors.forename: Angel T
authors.initials: AT
authors.lastname: Martínez
mesh_terms: D001665:Binding Sites; D003460:Crystallization; D018360:Crystallography, X-Ray; D005656:Fungal Proteins / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D008024:Ligands; D008345:Manganese / Q000737:chemistry / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010084:Oxidation-Reduction; D010544:Peroxidases / Q000737:chemistry* / Q000378:metabolism; D020076:Pleurotus / Q000201:enzymology* / Q000378:metabolism
chemical_list: D005656:Fungal Proteins; D008024:Ligands; D008345:Manganese; D010544:Peroxidases; C051129:manganese peroxidase