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 (lambda MP1) was constructed (R. E. Whitwam, I. G. Gazarian, and M. Tien, Biochem. Biophys. Res. Commun. 216, 1013-1017, 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 Km of recombinant Mn peroxidase for substrates H2O2 and the Mn2+/oxalate complex are 100 and 52 microM, respectively. The kcat as measured by Mn3+/oxalate formation is 450 s-1. 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 10(6) M-1 s-1, 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 10(4) M-1 s-1 is obtained for the recombinant enzyme. The reaction of compound II, the one-electron-oxidized state of the enzyme, with Mn2+/oxalate has a Kd of 13 microM and a first-order rate constant of 230 s-1 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.[1]References
- Heterologous expression and reconstitution of fungal Mn peroxidase. Whitwam, R., Tien, M. Arch. Biochem. Biophys. (1996) [Pubmed]
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