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Heterogeneity in the Histidine-brace Copper Coordination Sphere in Auxiliary Activity Family 10 (AA10) Lytic Polysaccharide Monooxygenases

DOI: 10.1074/jbc.M116.722447 DOI Help

Authors: Amanda Chaplin (University of Essex) , Michael T. Wilson (University of Essex) , Mike Hough (University of Essex) , Dimitri A. Svistunenko (School of Biological Sciences, University of Essex) , Glyn R. Hemsworth (Department of Chemistry, University of York, Diamond Light Source) , Paul Walton (University of York) , Erik Vijgenboom (Molecular Biotechnology, Institute of Biology, Sylvius Laboratory, Leiden University) , Jonathan Worrall (University of Essex)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Journal Of Biological Chemistry , VOL 291 , PAGES 12838 - 12850

State: Published (Approved)
Published: June 2016
Diamond Proposal Number(s): 7461

Abstract: Copper-dependent lytic polysaccharide monooxygenases (LPMOs) are enzymes that oxidatively deconstruct polysaccharides. The active site copper in LPMOs is coordinated by a histidine-brace. This utilizes the amino group and side chain of the N-terminal His residue with the side chain of a second His residue to create a T-shaped arrangement of nitrogen ligands. We report a structural, kinetic, and thermodynamic appraisal of copper binding to the histidine-brace in an auxiliary activity family 10 (AA10) LPMO from Streptomyces lividans (SliLPMO10E). Unexpectedly, we discovered the existence of two apo-SliLPMO10E species in solution that can each bind copper at a single site with distinct kinetic and thermodynamic (exothermic and endothermic) properties. The experimental EPR spectrum of copper-bound SliLPMO10E requires the simulation of two different line shapes, implying two different copper-bound species, indicative of three and two nitrogen ligands coordinating the copper. Amino group coordination was probed through the creation of an N-terminal extension variant (SliLPMO10E-Ext). The kinetics and thermodynamics of copper binding to SliLPMO10E-Ext are in accord with copper binding to one of the apo-forms in the wild-type protein, suggesting that amino group coordination is absent in the two-nitrogen coordinate form of SliLPMO10E. Copper binding to SliLPMO10B was also investigated, and again it revealed the presence of two apo-forms with kinetics and stoichiometry of copper binding identical to that of SliLPMO10E. Our findings highlight that heterogeneity exists in the active site copper coordination sphere of LPMOs that may have implications for the mechanism of loading copper in the cell.

Journal Keywords: copper monooxygenase; electron paramagnetic resonance (EPR); kinetics thermodynamics; x-ray crystallography

Subject Areas: Chemistry, Biology and Bio-materials, Medicine

Diamond Offline Facilities: JCSG Quality Control Server
Instruments: I03-Macromolecular Crystallography

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