title: Glycosylated linkers in multimodular lignocellulose-degrading enzymes dynamically bind to cellulose.
abstract: Plant cell-wall polysaccharides represent a vast source of food in nature. To depolymerize polysaccharides to soluble sugars, many organisms use multifunctional enzyme mixtures consisting of glycoside hydrolases, lytic polysaccharide mono-oxygenases, polysaccharide lyases, and carbohydrate esterases, as well as accessory, redox-active enzymes for lignin depolymerization. Many of these enzymes that degrade lignocellulose are multimodular with carbohydrate-binding modules (CBMs) and catalytic domains connected by flexible, glycosylated linkers. These linkers have long been thought to simply serve as a tether between structured domains or to act in an inchworm-like fashion during catalytic action. To examine linker function, we performed molecular dynamics (MD) simulations of the Trichoderma reesei Family 6 and Family 7 cellobiohydrolases (TrCel6A and TrCel7A, respectively) bound to cellulose. During these simulations, the glycosylated linkers bind directly to cellulose, suggesting a previously unknown role in enzyme action. The prediction from the MD simulations was examined experimentally by measuring the binding affinity of the Cel7A CBM and the natively glycosylated Cel7A CBM-linker. On crystalline cellulose, the glycosylated linker enhances the binding affinity over the CBM alone by an order of magnitude. The MD simulations before and after binding of the linker also suggest that the bound linker may affect enzyme action due to significant damping in the enzyme fluctuations. Together, these results suggest that glycosylated linkers in carbohydrate-active enzymes, which are intrinsically disordered proteins in solution, aid in dynamic binding during the enzymatic deconstruction of plant cell walls.
authors: Biosciences Center and National Bioenergy Center, National Renewable Energy Laboratory, Golden, CO 80401, USA
authors: Christina M
authors: CM
authors: Payne
authors: Michael G
authors: MG
authors: Resch
authors: Liqun
authors: L
authors: Chen
authors: Michael F
authors: MF
authors: Crowley
authors: Michael E
authors: ME
authors: Himmel
authors: Larry E
authors: LE
authors: Taylor
authors: Mats
authors: M
authors: Sandgren
authors: Jerry
authors: J
authors: Ståhlberg
authors: Ingeborg
authors: I
authors: Stals
authors: Zhongping
authors: Z
authors: Tan
authors: Gregg T
authors: GT
authors: Beckham
mesh_terms: D001665:Binding Sites; D001667:Binding, Competitive; D055162:Biocatalysis; D020134:Catalytic Domain; D002482:Cellulose / Q000378:metabolism*; D043366:Cellulose 1,4-beta-Cellobiosidase / Q000737:chemistry / Q000378:metabolism*; D005656:Fungal Proteins / Q000737:chemistry / Q000378:metabolism*; D006031:Glycosylation; D006868:Hydrolysis; D008031:Lignin / Q000378:metabolism*; D013058:Mass Spectrometry; D008958:Models, Molecular; D056004:Molecular Dynamics Simulation; D011485:Protein Binding; D014242:Trichoderma / Q000201:enzymology / Q000378:metabolism
chemical_list: D005656:Fungal Proteins; C036909:lignocellulose; D002482:Cellulose; D008031:Lignin; D043366:Cellulose 1,4-beta-Cellobiosidase
keywords: biofuels; carbohydrate recognition; cellulase; post-translational modification
references: Biochemistry. 1989 Sep 5;28(18):7241-57
references: 2554967
references: Science. 1994 Jul 22;265(5171):524-8
references: 8036495
references: J Comput Chem. 2004 Aug;25(11):1400-15
references: 15185334
references: J Chem Theory Comput. 2009 Aug 20;5(9):2353-2370
references: 20161005
references: Nucleic Acids Res. 2009 Jan;37(Database issue):D233-8
references: 18838391
references: Glycobiology. 2004 Aug;14(8):713-24
references: 15070858
references: J Biol Chem. 2012 Oct 19;287(43):36322-30
references: 22952223
references: J Biol Chem. 2007 Jan 19;282(3):2091-100
references: 17121820
references: Microbiol Mol Biol Rev. 2005 Mar;69(1):124-54
references: 15755956
references: J Chem Theory Comput. 2011 Oct 11;7(10):3162-3180
references: 22125473
references: Science. 2007 Feb 9;315(5813):804-7
references: 17289988
references: Biochem J. 2010 Dec 15;432(3):437-44
references: 20925655
references: Proc Natl Acad Sci U S A. 2011 Sep 13;108(37):15079-84
references: 21876164
references: Structure. 2011 Jul 13;19(7):907-17
references: 21742258
references: Proc Natl Acad Sci U S A. 2010 Aug 24;107(34):15293-8
references: 20696902
references: Proc Natl Acad Sci U S A. 2000 May 23;97(11):5842-7
references: 10823940
references: Science. 2012 Jun 29;336(6089):1715-9
references: 22745431
references: Structure. 1999 Sep 15;7(9):1035-45
references: 10508787
references: FEBS Lett. 1998 Jun 16;429(3):341-6
references: 9662445
references: Biochemistry. 1996 Jan 16;35(2):586-92
references: 8555231
references: J Biol Chem. 2013 May 10;288(19):13503-10
references: 23532843
references: J Mol Biol. 1998 Jan 16;275(2):309-25
references: 9466911
references: J Biol Chem. 1993 Oct 5;268(28):20756-61
references: 8407900
references: Annu Rev Microbiol. 2004;58:521-54
references: 15487947
references: Microbiol Mol Biol Rev. 2002 Sep;66(3):506-77, table of contents
references: 12209002
references: Proc Natl Acad Sci U S A. 2003 Nov 25;100(24):13809-14
references: 14623971
references: J Comput Chem. 2005 Dec;26(16):1781-802
references: 16222654
references: J Comput Chem. 2009 Jul 30;30(10):1545-614
references: 19444816
references: Proc Natl Acad Sci U S A. 2003 Jan 21;100(2):484-9
references: 12522267
references: Glycobiology. 2008 Aug;18(8):626-37
references: 18504293
references: J Biol Chem. 2002 Oct 25;277(43):40887-92
references: 12186865
references: J Phys Chem B. 2011 Apr 14;115(14):4118-27
references: 21425804
references: Biophys J. 2010 Dec 1;99(11):3773-81
references: 21112302
references: J Chromatogr B Biomed Sci Appl. 2001 Mar 10;752(2):349-68
references: 11270873
references: J Am Chem Soc. 2012 Jan 18;134(2):890-2
references: 22188218
references: J Biol Chem. 2011 Nov 25;286(47):41028-35
references: 21965672
references: J Bacteriol. 1998 Jun;180(12):3091-9
references: 9620957
references: Bioinformatics. 2006 Jan 15;22(2):195-201
references: 16301204
references: J Biol Chem. 2009 Dec 25;284(52):36186-36190
references: 19858200
references: PLoS One. 2012;7(11):e48615
references: 23139804
references: Biochem J. 2004 Sep 15;382(Pt 3):769-81
references: 15214846
references: Science. 2011 Sep 2;333(6047):1279-82
references: 21885779
references: J Biol Chem. 2012 Aug 3;287(32):26953-61
references: 22707718
references: J Am Chem Soc. 2002 Aug 7;124(31):9074-82
references: 12149011
references: Curr Opin Struct Biol. 2005 Dec;15(6):637-45
references: 16263268
references: Biotechnol Biofuels. 2013 Mar 21;6(1):41
references: 23514094
references: J Biol Chem. 1991 Jun 15;266(17):11335-40
references: 1904063
references: Annu Rev Microbiol. 1987;41:465-505
references: 3318677
references: Protein Sci. 1995 Jun;4(6):1056-64
references: 7549870
references: J Mol Graph Model. 2001;19(1):26-59
references: 11381529
references: J Phys Chem B. 2009 Apr 9;113(14):4970-7
references: 19292431
references: Eur J Biochem. 1998 Aug 15;256(1):119-27
references: 9746354
references: Science. 2010 Oct 8;330(6001):219-22
references: 20929773