Crystal Structures of Substrate Binding Site Mutants of Manganese Peroxidase

Munirathinam Sundaramoorthy; Katsuyuki Kishi; Michael H. Gold; Thomas L. Poulos

doi:10.1074/jbc.272.28.17574

Crystal Structures of Substrate Binding Site Mutants of Manganese Peroxidase*

From the ^‡Department of Molecular Biology & Biochemistry and Physiology & Biophysics, University of California, Irvine, California 92697-3900 and ^§Department of Chemistry, Biochemistry, and Molecular Biology, Oregon Graduate Institute of Science & Technology, Portland, Oregon 97291-1000

Abstract

Manganese peroxidase (MnP), an extracellular heme enzyme from the lignin-degrading basidiomycetous fungus,Phanerochaete chrysosporium, catalyzes the oxidation of Mn^II to Mn^III. The latter, acting as a diffusible redox mediator, is capable of oxidizing a variety of lignin model compounds. The proposed Mn^II binding site of MnP consists of a heme propionate, three acidic ligands (Glu-35, Glu-39, and Asp-179), and two water molecules. Using crystallographic methods, this binding site was probed by altering the amount of Mn^IIbound to the protein. Crystals grown in the absence of Mn^II, or in the presence of EDTA, exhibited diminished electron density at this site. Crystals grown in excess Mn^II exhibited increased electron density at the proposed binding site but nowhere else in the protein. This suggests that there is only one major Mn^II binding site in MnP. Crystal structures of a single mutant (D179N) and a double mutant (E35Q,D179N) at this site were determined. The mutant structures lack a cation at the Mn^II binding site. The structure of the Mn^II binding site is altered significantly in both mutants, resulting in increased access to the solvent and substrate.

Footnotes

↵* This research is supported by National Science Foundation Grants MCB-9405128 (to T. L. P.) and MCB-9405978 (to M. H. G.) and by U.S. Department of Energy, Division of Energy Biosciences Grant DE-FG06-93ER20093 (to M. H. G.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The atomic coordinates and structure of the MnP1 crystal structure (code 1MNP) have been deposited in the Protein Data Bank, Brookhaven National Laboratory, Upton, NY.
↵¶ To whom correspondence should be addressed: Dept. of Molecular Biology & Biochemistry, University of California, Irvine, CA 92697-3900. Tel.: 714-824-7020; Fax: 714-824-3280; E-mail:poulos{at}uci.edu.