title: The copper active site of CBM33 polysaccharide oxygenases.
abstract: The capacity of metal-dependent fungal and bacterial polysaccharide oxygenases, termed GH61 and CBM33, respectively, to potentiate the enzymatic degradation of cellulose opens new possibilities for the conversion of recalcitrant biomass to biofuels. GH61s have already been shown to be unique metalloenzymes containing an active site with a mononuclear copper ion coordinated by two histidines, one of which is an unusual τ-N-methylated N-terminal histidine. We now report the structural and spectroscopic characterization of the corresponding copper CBM33 enzymes. CBM33 binds copper with high affinity at a mononuclear site, significantly stabilizing the enzyme. X-band EPR spectroscopy of Cu(II)-CBM33 shows a mononuclear type 2 copper site with the copper ion in a distorted axial coordination sphere, into which azide will coordinate as evidenced by the concomitant formation of a new absorption band in the UV/vis spectrum at 390 nm. The enzyme's three-dimensional structure contains copper, which has been photoreduced to Cu(I) by the incident X-rays, confirmed by X-ray absorption/fluorescence studies of both aqueous solution and intact crystals of Cu-CBM33. The single copper(I) ion is ligated in a T-shaped configuration by three nitrogen atoms from two histidine side chains and the amino terminus, similar to the endogenous copper coordination geometry found in fungal GH61.
authors.affiliation: Department of Chemistry, University of York, Heslington, York YO10 5DD, United Kingdom.
authors.forename: Glyn R
authors.initials: GR
authors.lastname: Hemsworth
authors.forename: Edward J
authors.initials: EJ
authors.lastname: Taylor
authors.forename: Robbert Q
authors.initials: RQ
authors.lastname: Kim
authors.forename: Rebecca C
authors.initials: RC
authors.lastname: Gregory
authors.forename: Sally J
authors.initials: SJ
authors.lastname: Lewis
authors.forename: Johan P
authors.initials: JP
authors.lastname: Turkenburg
authors.forename: Alison
authors.initials: A
authors.lastname: Parkin
authors.forename: Gideon J
authors.initials: GJ
authors.lastname: Davies
authors.forename: Paul H
authors.initials: PH
authors.lastname: Walton
mesh_terms: D001407:Bacillus / Q000201:enzymology; D002151:Calorimetry; D020134:Catalytic Domain; D003300:Copper / Q000737:chemistry*; D004578:Electron Spin Resonance Spectroscopy; D005470:Fluorometry; D006639:Histidine / Q000737:chemistry; D009682:Magnetic Resonance Spectroscopy; D008667:Metalloproteins / Q000737:chemistry*; D008670:Metals / Q000737:chemistry; D008958:Models, Molecular; D010084:Oxidation-Reduction; D010105:Oxygenases / Q000737:chemistry*; D011487:Protein Conformation; D013056:Spectrophotometry, Ultraviolet; D014961:X-Ray Diffraction
chemical_list: D008667:Metalloproteins; D008670:Metals; D006639:Histidine; D003300:Copper; D010105:Oxygenases
references.citation: Anal Biochem. 2013 Feb 1;433(1):43-7
references.pmid: 23098701
references.citation: Science. 2012 Jun 29;336(6089):1715-9
references.pmid: 22745431
references.citation: Biotechnol Biofuels. 2012 Jul 02;5(1):45
references.pmid: 22747961
references.citation: Biochem J. 2004 Sep 15;382(Pt 3):769-81
references.pmid: 15214846
references.citation: Biochemistry. 2011 Nov 29;50(47):10231-40
references.pmid: 22013879
references.citation: J Am Chem Soc. 2007 May 2;129(17):5352-3
references.pmid: 17411054
references.citation: Biochemistry. 2010 Apr 20;49(15):3305-16
references.pmid: 20230050
references.citation: Appl Environ Microbiol. 2012 Sep;78(17):6161-71
references.pmid: 22729546
references.citation: Acta Crystallogr D Biol Crystallogr. 2005 Apr;61(Pt 4):458-64
references.pmid: 15805601
references.citation: Nature. 2013 Jan 24;493(7433):514-7
references.pmid: 23334409
references.citation: Protein Sci. 2011 Sep;20(9):1479-83
references.pmid: 21748815
references.citation: Microbiology (Reading). 2001 Jul;147(Pt 7):1793-1803
references.pmid: 11429457
references.citation: Science. 2004 May 7;304(5672):864-7
references.pmid: 15131304
references.citation: Acta Crystallogr D Biol Crystallogr. 2011 Apr;67(Pt 4):235-42
references.pmid: 21460441
references.citation: Appl Environ Microbiol. 2011 Oct;77(19):7007-15
references.pmid: 21821740
references.citation: Angew Chem Int Ed Engl. 2008;47(47):9084-7
references.pmid: 18932185
references.citation: Proc Natl Acad Sci U S A. 2011 Sep 13;108(37):15079-84
references.pmid: 21876164
references.citation: Acta Crystallogr D Biol Crystallogr. 2004 Dec;60(Pt 12 Pt 1):2288-94
references.pmid: 15572783
references.citation: Angew Chem Int Ed Engl. 2009;48(43):8086-9
references.pmid: 19784981
references.citation: Acta Crystallogr D Biol Crystallogr. 1997 May 1;53(Pt 3):240-55
references.pmid: 15299926
references.citation: Appl Microbiol Biotechnol. 2012 Aug;95(4):979-90
references.pmid: 22718248
references.citation: Structure. 2012 Jun 6;20(6):1051-61
references.pmid: 22578542
references.citation: J Biol Chem. 2005 Mar 25;280(12):11313-9
references.pmid: 15590674
references.citation: Appl Environ Microbiol. 2013 Jan;79(2):488-96
references.pmid: 23124232
references.citation: PLoS One. 2011;6(11):e27807
references.pmid: 22132148
references.citation: Proc Natl Acad Sci U S A. 2012 Nov 13;109(46):18779-84
references.pmid: 23112164
references.citation: Angew Chem Int Ed Engl. 2010 Feb 15;49(8):1359-61
references.pmid: 20013977
references.citation: Nucleic Acids Res. 2009 Jan;37(Database issue):D233-8
references.pmid: 18838391
references.citation: J Mol Biol. 2012 Feb 17;416(2):239-54
references.pmid: 22210154
references.citation: Nat Protoc. 2008;3(7):1171-9
references.pmid: 18600222
references.citation: Acc Chem Res. 2007 Jul;40(7):601-8
references.pmid: 17458929
references.citation: ACS Chem Biol. 2011 Dec 16;6(12):1399-406
references.pmid: 22004347
references.citation: Biochemistry. 1972 May 23;11(11):2187-92
references.pmid: 4337491
references.citation: Inorg Chem. 2005 Feb 21;44(4):770-8
references.pmid: 15859245
references.citation: J Am Chem Soc. 2012 Jan 18;134(2):890-2
references.pmid: 22188218
references.citation: Arch Biochem Biophys. 1974 Dec;165(2):691-708
references.pmid: 4374138
references.citation: Nat Chem Biol. 2013 Mar;9(3):177-83
references.pmid: 23354287
references.citation: FEBS J. 2013 Jul;280(13):3028-49
references.pmid: 23398882
references.citation: Acta Crystallogr D Biol Crystallogr. 2010 Feb;66(Pt 2):125-32
references.pmid: 20124692
references.citation: Methods Enzymol. 2012;510:69-95
references.pmid: 22608722
references.citation: Appl Microbiol Biotechnol. 2012 Mar;93(5):2075-89
references.pmid: 22290653
references.citation: Science. 2010 Oct 8;330(6001):219-22
references.pmid: 20929773