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 expresse d in Escherichia coli. A bacterial expression vector for the cDNA of M n peroxidase isozyme H4 (lambda MP1) was constructed (R. E. Whitwam, I . G. Gazarian, and M. Tien, Biochem. Biophys. Res. Commun. 216, 1013-1 017, 1995) whose expression in E. coli results in the formation of cat alytically 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 p resence of CaCl2, hemin, and oxidized glutathione. The recombinant enz yme had the same spectral and kinetic properties as the native fungal enzyme, The K-m of recombinant Mn peroxidase for substrates H2O2 and t he Mn2+/oxalate complex are 100 and 52 mu M, respectively. The k(cat) as measured by Mn3+/oxalate formation is 450 s(-1). These are essentia lly the same values as seen with the native fungal enzyme, The rate of formation of compound I, the two-electron-oxidized state of the enzym e, is 4.0 x 10(6) M(-1) s(-1), identical to the rate of the native fun gal 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 p H of 2.5 a rate of 4.2 x 10(4) M(-1) s(-1) is obtained for the recombi nant enzyme, The reaction of compound II, the one-electron-oxidized st ate of the enzyme, with Mn2+/oxalate has a K-d of 13 mu M and a first- order rate constant of 230 s(-1) in the recombinant enzyme, These rate s are essentially the same as those seen with the native fungal MnP. T hese results demonstrate that the bacterial expression of recombinant Mn peroxidase is a convenient and efficient system for the expression and characterization of Mn peroxidase. (C) 1996 Academic Press, Inc.