title: Engineering a disulfide bond in recombinant manganese peroxidase results in increased thermostability.
abstract: Manganese peroxidase (MnP) produced by Phanerochaete chrysosporium, which catalyzes the oxidation of Mn(2+) to Mn(3+) by hydrogen peroxide, was shown to be susceptible to thermal inactivation due to the loss of calcium [Sutherland, G. R. J.; Aust, S. D. Arch. Biochem. Biophys. 1996, 332, 128-134]. The recombinant enzyme, lacking glycosylation, was found to be more susceptible [Nie, G.; Reading, N. S.; Aust, S. D. Arch. Biochem. Biophys. 1999, 365, 328-334]. On the basis of the properties and structure of peanut peroxidase, we have engineered a disulfide bond near the distal calcium binding site of MnP by means of the double mutation A48C and A63C. The mutant enzyme had activity and spectral properties similar to those of native, glycosylated MnP. The thermostabilities of native, recombinant, and mutant MnP were studied as a function of temperature and pH. MnPA48C/A63C exhibited kinetics of inactivation similar to that of native MnP. The addition of calcium decreased the rate of thermal inactivation of the enzymes, while EGTA increased the rate of inactivation. Thermally treated MnPA48C/A63C mutant was shown to contain one calcium, and it retained a percentage of its original manganese oxidase activity; native and recombinant MnP were inactivated by the removal of calcium from the protein.
authors.affiliation: Biotechnology Center, Utah State University, Logan, Utah 84322-4705, USA.
authors.forename: N S
authors.initials: NS
authors.lastname: Reading
authors.forename: S D
authors.initials: SD
authors.lastname: Aust
mesh_terms: D001483:Base Sequence; D002118:Calcium / Q000378:metabolism; D004247:DNA; D004795:Enzyme Stability; D016297:Mutagenesis, Site-Directed; D010544:Peroxidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013696:Temperature
chemical_list: D011994:Recombinant Proteins; D004247:DNA; D010544:Peroxidases; C051129:manganese peroxidase; D002118:Calcium