title: Sequencing, biochemical characterization, crystal structure and molecular dynamics of cellobiohydrolase Cel7A from Geotrichum candidum 3C.
abstract: The ascomycete Geotrichum candidum is a versatile and efficient decay fungus that is involved, for example, in biodeterioration of compact discs; notably, the 3C strain was previously shown to degrade filter paper and cotton more efficiently than several industrial enzyme preparations. Glycoside hydrolase (GH) family 7 cellobiohydrolases (CBHs) are the primary constituents of industrial cellulase cocktails employed in biomass conversion, and feature tunnel-enclosed active sites that enable processive hydrolytic cleavage of cellulose chains. Understanding the structure-function relationships defining the activity and stability of GH7 CBHs is thus of keen interest. Accordingly, we report the comprehensive characterization of the GH7 CBH secreted by G. candidum (GcaCel7A). The bimodular cellulase consists of a family 1 cellulose-binding module (CBM) and linker connected to a GH7 catalytic domain that shares 64% sequence identity with the archetypal industrial GH7 CBH of Hypocrea jecorina (HjeCel7A). GcaCel7A shows activity on Avicel cellulose similar to HjeCel7A, with less product inhibition, but has a lower temperature optimum (50 °C versus 60-65 °C, respectively). Five crystal structures, with and without bound thio-oligosaccharides, show conformational diversity of tunnel-enclosing loops, including a form with partial tunnel collapse at subsite -4 not reported previously in GH7. Also, the first O-glycosylation site in a GH7 crystal structure is reported--on a loop where the glycan probably influences loop contacts across the active site and interactions with the cellulose surface. The GcaCel7A structures indicate higher loop flexibility than HjeCel7A, in accordance with sequence modifications. However, GcaCel7A retains small fluctuations in molecular simulations, suggesting high processivity and low endo-initiation probability, similar to HjeCel7A.
  
  
  METHODS
  Structural data are available in the Protein Data Bank under the accession numbers 5AMP, 4ZZV, 4ZZW, 4ZZT, and 4ZZU. The Geotrichum candidum GH family 7 cellobiohydrolase nucleotide sequence is available in GenBank under accession number KJ958925.
  Glycoside hydrolase family 7 reducing end acting cellobiohydrolase.
authors.affiliation: Department of Chemistry and Biotechnology, Swedish University of Agricultural Sciences, Uppsala, Sweden.|National Research Centre «Kurchatov Institute», B.P. Konstantinov Petersburg Nuclear Physics Institute, Gatchina, Orlova roscha, Russia.
authors.forename: Anna S
authors.initials: AS
authors.lastname: Borisova
authors.affiliation: National Research Centre «Kurchatov Institute», B.P. Konstantinov Petersburg Nuclear Physics Institute, Gatchina, Orlova roscha, Russia.
authors.forename: Elena V
authors.initials: EV
authors.lastname: Eneyskaya
authors.affiliation: National Research Centre «Kurchatov Institute», B.P. Konstantinov Petersburg Nuclear Physics Institute, Gatchina, Orlova roscha, Russia.
authors.forename: Kirill S
authors.initials: KS
authors.lastname: Bobrov
authors.affiliation: Department of Chemical and Materials Engineering, University of Kentucky, Lexington, KY, USA.
authors.forename: Suvamay
authors.initials: S
authors.lastname: Jana
authors.affiliation: Theodosius Dobzhansky Center for Genome Bioinformatics, St. Petersburg State University, Russia.
authors.forename: Anton
authors.initials: A
authors.lastname: Logachev
authors.affiliation: Research Resource Centre «Molecular and Cell Technologies», St. Petersburg State University, Russia.
authors.forename: Dmitrii E
authors.initials: DE
authors.lastname: Polev
authors.affiliation: Centre for Algorithmic Biotechnology, St. Petersburg Academic University, Russia.
authors.forename: Alla L
authors.initials: AL
authors.lastname: Lapidus
authors.affiliation: National Research Centre «Kurchatov Institute», B.P. Konstantinov Petersburg Nuclear Physics Institute, Gatchina, Orlova roscha, Russia.
authors.forename: Farid M
authors.initials: FM
authors.lastname: Ibatullin
authors.affiliation: Department of Chemistry and Biotechnology, Swedish University of Agricultural Sciences, Uppsala, Sweden.
authors.forename: Umair
authors.initials: U
authors.lastname: Saleem
authors.affiliation: Department of Chemistry and Biotechnology, Swedish University of Agricultural Sciences, Uppsala, Sweden.
authors.forename: Mats
authors.initials: M
authors.lastname: Sandgren
authors.affiliation: Department of Chemical and Materials Engineering, University of Kentucky, Lexington, KY, USA.
authors.forename: Christina M
authors.initials: CM
authors.lastname: Payne
authors.affiliation: National Research Centre «Kurchatov Institute», B.P. Konstantinov Petersburg Nuclear Physics Institute, Gatchina, Orlova roscha, Russia.|Department of Medical Physics, Peter the Great St. Petersburg Polytechnic University, Russia.
authors.forename: Anna A
authors.initials: AA
authors.lastname: Kulminskaya
authors.affiliation: Department of Chemistry and Biotechnology, Swedish University of Agricultural Sciences, Uppsala, Sweden.
authors.forename: Jerry
authors.identifier: http://orcid.org/0000-0003-4059-8580
authors.initials: J
authors.lastname: Ståhlberg
mesh_terms: D000595:Amino Acid Sequence; D043366:Cellulose 1,4-beta-Cellobiosidase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D005848:Geotrichum / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D056004:Molecular Dynamics Simulation; D008969:Molecular Sequence Data; D011487:Protein Conformation; D016415:Sequence Alignment; D013696:Temperature
chemical_list: D043366:Cellulose 1,4-beta-Cellobiosidase
keywords: Geotrichum candidum; X-ray structure; biomass degradation; cellulase; molecular dynamics