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J Biol Chem. 2014 Jul 4;289(27):18782-92. doi: 10.1074/jbc.M114.563494. Epub 2014 May 14.

Structural and electronic snapshots during the transition from a Cu(II) to Cu(I) metal center of a lytic polysaccharide monooxygenase by X-ray photoreduction.

Author information

1
From the Department of Chemistry and Biotechnology, Swedish University of Agricultural Sciences, SE-750 07 Uppsala, Sweden.
2
the National Bioenergy Center, National Renewable Energy Laboratory, Golden, Colorado 80401.
3
From the Department of Chemistry and Biotechnology, Swedish University of Agricultural Sciences, SE-750 07 Uppsala, Sweden, the Department of Biomaterials Sciences, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo 113-8657, Japan.
4
the Department of Chemistry, Biotechnology and Food Science, Norwegian University of Life Sciences, NO-1432 Ås, Norway.
5
the Institut de Biologie Structurale, Université Grenoble Alpes-CNRS-CEA, 38042 Grenoble, France, and the European Synchrotron Radiation Facility, 38000 Grenoble, France.
6
From the Department of Chemistry and Biotechnology, Swedish University of Agricultural Sciences, SE-750 07 Uppsala, Sweden, the Department of Chemistry, Biotechnology and Food Science, Norwegian University of Life Sciences, NO-1432 Ås, Norway.
7
the National Bioenergy Center, National Renewable Energy Laboratory, Golden, Colorado 80401, gregg.beckham@nrel.gov.
8
From the Department of Chemistry and Biotechnology, Swedish University of Agricultural Sciences, SE-750 07 Uppsala, Sweden, mats.sandgren@slu.se.

Abstract

Lytic polysaccharide monooxygenases (LPMOs) are a recently discovered class of enzymes that employ a copper-mediated, oxidative mechanism to cleave glycosidic bonds. The LPMO catalytic mechanism likely requires that molecular oxygen first binds to Cu(I), but the oxidation state in many reported LPMO structures is ambiguous, and the changes in the LPMO active site required to accommodate both oxidation states of copper have not been fully elucidated. Here, a diffraction data collection strategy minimizing the deposited x-ray dose was used to solve the crystal structure of a chitin-specific LPMO from Enterococcus faecalis (EfaCBM33A) in the Cu(II)-bound form. Subsequently, the crystalline protein was photoreduced in the x-ray beam, which revealed structural changes associated with the conversion from the initial Cu(II)-oxidized form with two coordinated water molecules, which adopts a trigonal bipyramidal geometry, to a reduced Cu(I) form in a T-shaped geometry with no coordinated water molecules. A comprehensive survey of Cu(II) and Cu(I) structures in the Cambridge Structural Database unambiguously shows that the geometries observed in the least and most reduced structures reflect binding of Cu(II) and Cu(I), respectively. Quantum mechanical calculations of the oxidized and reduced active sites reveal little change in the electronic structure of the active site measured by the active site partial charges. Together with a previous theoretical investigation of a fungal LPMO, this suggests significant functional plasticity in LPMO active sites. Overall, this study provides molecular snapshots along the reduction process to activate the LPMO catalytic machinery and provides a general method for solving LPMO structures in both copper oxidation states.

KEYWORDS:

Carbohydrate-binding Protein; Cellulase; Metalloenzyme; Polysaccharide; X-ray Crystallography

PMID:
24828494
PMCID:
PMC4081921
DOI:
10.1074/jbc.M114.563494
[Indexed for MEDLINE]
Free PMC Article

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