Heterologous expression and reconstitution of fungal Mn peroxidase

Ross Whitwam, Ming Tien

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Abstract

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.

Original languageEnglish (US)
Pages (from-to)439-446
Number of pages8
JournalArchives of Biochemistry and Biophysics
Volume333
Issue number2
DOIs
Publication statusPublished - Sep 15 1996

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All Science Journal Classification (ASJC) codes

  • Biophysics
  • Biochemistry
  • Molecular Biology

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