title: Anomeric Selectivity and Product Profile of a Processive Cellulase.
abstract: Cellobiohydrolases (CBHs) make up an important group of enzymes for both natural carbon cycling and industrial deconstruction of lignocellulosic biomass. The consecutive hydrolysis of one cellulose strand relies on an intricate pattern of enzyme-substrate interactions in the long, tunnel-shaped binding site of the CBH. In this work, we have investigated the initial complexation mode with cellulose of the most thoroughly studied CBH, Cel7A from Hypocrea jecorina (HjCel7A). We found that HjCel7A predominantly produces glucose when it initiates a processive run on insoluble microcrystalline cellulose, confirming the validity of an even and odd product ratio as an estimate of processivity. Moreover, the glucose released from cellulose was predominantly α-glucose. A link between the initial binding mode of the enzyme and the reducing end configuration was investigated by inhibition studies with the two anomers of cellobiose. A clear preference for β-cellobiose in product binding site +2 was observed for HjCel7A, but not the homologous endoglucanase, HjCe7B. Possible relationships between this anomeric preference in the product site and the prevalence of odd-numbered initial-cut products are discussed, and a correlation between processivity and anomer selectivity is proposed.
authors.affiliation: Research Unit for Functional Biomaterials, Roskilde University , Roskilde, Denmark.
authors.forename: Jeppe
authors.initials: J
authors.lastname: Kari
authors.affiliation: Institute of Molecular and Cell Biology, University of Tartu , Tartu, Estonia.
authors.forename: Riin
authors.initials: R
authors.lastname: Kont
authors.affiliation: Novozymes A/S , Krogshøjvej 36, DK-2880 Bagsværd, Denmark.
authors.forename: Kim
authors.initials: K
authors.lastname: Borch
authors.affiliation: Novozymes A/S , Krogshøjvej 36, DK-2880 Bagsværd, Denmark.
authors.forename: Steen
authors.initials: S
authors.lastname: Buskov
authors.affiliation: Research Unit for Functional Biomaterials, Roskilde University , Roskilde, Denmark.
authors.forename: Johan Pelck
authors.initials: JP
authors.lastname: Olsen
authors.affiliation: Novozymes A/S , Krogshøjvej 36, DK-2880 Bagsværd, Denmark.
authors.forename: Nicolaj
authors.initials: N
authors.lastname: Cruyz-Bagger
authors.affiliation: Institute of Molecular and Cell Biology, University of Tartu , Tartu, Estonia.
authors.forename: Priit
authors.initials: P
authors.lastname: Väljamäe
authors.affiliation: Research Unit for Functional Biomaterials, Roskilde University , Roskilde, Denmark.
authors.forename: Peter
authors.initials: P
authors.lastname: Westh
mesh_terms: D000465:Algorithms; D015374:Biosensing Techniques; D002475:Cellobiose / Q000737:chemistry / Q000378:metabolism*; D002482:Cellulose / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D043366:Cellulose 1,4-beta-Cellobiosidase / Q000737:chemistry / Q000378:metabolism*; D002853:Chromatography, Liquid; D018360:Crystallography, X-Ray; D005656:Fungal Proteins / Q000737:chemistry / Q000378:metabolism*; D005947:Glucose / Q000737:chemistry / Q000378:metabolism; D035901:Hypocrea / Q000201:enzymology* / Q000378:metabolism; D007527:Isoenzymes / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D013058:Mass Spectrometry; D008958:Models, Molecular; D015394:Molecular Structure; D011485:Protein Binding; D000072417:Protein Domains; D013379:Substrate Specificity; D013780:Tetroses / Q000737:chemistry / Q000378:metabolism
chemical_list: D005656:Fungal Proteins; D007527:Isoenzymes; D013780:Tetroses; D002475:Cellobiose; C048468:cellotetraose; D002482:Cellulose; D043366:Cellulose 1,4-beta-Cellobiosidase; D005947:Glucose