Heterologous expression and reconstitution of fungal Mn peroxidase

We have optimized the conditions under which recombinant Mn peroxidase from the white-rot fungus Phanerochaete chrysosporium can be expressed in Escherichia coli. A bacterial expression vector for the cDNA of Mn peroxidase isozyme H4 (λMP1) was constructed (R. E. Whitwam, I. G. Gazarian, and M. Tien, Biochem. Biophys. Res. Commun. 216, 10131017, 1995) whose expression in E. coli results in the formation of catalytically inactive polypeptide which can be refolded to active enzyme. The refolded enzyme was purified to homogeneity. Refolding was most efficient in 2 M urea, pH 8.0, and was absolutely dependent upon the presence of CaCl₂, hemin, and oxidized glutathione. The recombinant enzyme had the same spectral and kinetic properties as the native fungal enzyme. The K(m) of recombinant Mn peroxidase for substrates H²O₂ and the Mn²⁺/oxalate complex are 100 and 52 μM, respectively. The k(cat) as measured by Mn³⁺/oxalate formation is 450 s¹. These are essentially the same values as seen with the native fungal enzyme. The rate of formation of compound I, the two-electron-oxidized state of the enzyme, is 4.0 x 10⁶ M¹ s¹, identical to the rate of the native fungal Mn peroxidase. The reaction of compound I with Mn²⁺ is too fast to measure at pH 4.5 in the recombinant Mn peroxidase. At a suboptimal pH of 2.5 a rate of 4.2 x 10⁴ M¹ s¹ is obtained for the recombinant enzyme. The reaction of compound II, the one-electron-oxidized state of the enzyme, with Mn²⁺/oxalate has a K(d) of 13 μM and a first-order rate constant of 230 s¹ in the recombinant enzyme. These rates are essentially the same as those seen with the native fungal MnP. These results demonstrate that the bacterial expression of recombinant Mn peroxidase is a convenient and efficient system for the expression and characterization of Mn peroxidase.