title: Heterogeneity in the Histidine-brace Copper Coordination Sphere in Auxiliary Activity Family 10 (AA10) Lytic Polysaccharide Monooxygenases.
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.
authors.affiliation: From the School of Biological Sciences, University of Essex, Wivenhoe Park, Colchester CO4 3SQ, United Kingdom.
authors.forename: Amanda K
authors.initials: AK
authors.lastname: Chaplin
authors.affiliation: From the School of Biological Sciences, University of Essex, Wivenhoe Park, Colchester CO4 3SQ, United Kingdom.
authors.forename: Michael T
authors.initials: MT
authors.lastname: Wilson
authors.affiliation: From the School of Biological Sciences, University of Essex, Wivenhoe Park, Colchester CO4 3SQ, United Kingdom.
authors.forename: Michael A
authors.initials: MA
authors.lastname: Hough
authors.affiliation: From the School of Biological Sciences, University of Essex, Wivenhoe Park, Colchester CO4 3SQ, United Kingdom.
authors.forename: Dimitri A
authors.initials: DA
authors.lastname: Svistunenko
authors.affiliation: the Department of Chemistry, University of York, Heslington, York YO10 5DD, United Kingdom, and.
authors.forename: Glyn R
authors.initials: GR
authors.lastname: Hemsworth
authors.affiliation: the Department of Chemistry, University of York, Heslington, York YO10 5DD, United Kingdom, and.
authors.forename: Paul H
authors.initials: PH
authors.lastname: Walton
authors.affiliation: the Molecular Biotechnology, Institute of Biology, Sylvius Laboratory, Leiden University, P. O. Box 9505, 2300RA Leiden, The Netherlands.
authors.forename: Erik
authors.initials: E
authors.lastname: Vijgenboom
authors.affiliation: From the School of Biological Sciences, University of Essex, Wivenhoe Park, Colchester CO4 3SQ, United Kingdom,. Electronic address: jworrall@essex.ac.uk.
authors.forename: Jonathan A R
authors.initials: JAR
authors.lastname: Worrall
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020134:Catalytic Domain; D003300:Copper / Q000737:chemistry* / Q000378:metabolism; D018360:Crystallography, X-Ray; D004578:Electron Spin Resonance Spectroscopy; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D006639:Histidine / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D013058:Mass Spectrometry; D006899:Mixed Function Oxygenases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D009154:Mutation; D009942:Organometallic Compounds / Q000737:chemistry* / Q000378:metabolism; D011134:Polysaccharides / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D013050:Spectrometry, Fluorescence; D048170:Streptomyces lividans / Q000201:enzymology / Q000235:genetics; D013379:Substrate Specificity; D013816:Thermodynamics
chemical_list: D001426:Bacterial Proteins; D009942:Organometallic Compounds; D011134:Polysaccharides; D006639:Histidine; D003300:Copper; D006899:Mixed Function Oxygenases
keywords: copper monooxygenase; electron paramagnetic resonance (EPR); kinetics; thermodynamics; x-ray crystallography