title: Cellobiohydrolase 1 from Trichoderma reesei degrades cellulose in single cellobiose steps.
abstract: Cellobiohydrolase 1 from Trichoderma reesei (TrCel7A) processively hydrolyses cellulose into cellobiose. Although enzymatic techniques have been established as promising tools in biofuel production, a clear understanding of the motor's mechanistic action has yet to be revealed. Here, we develop an optical tweezers-based single-molecule (SM) motility assay for precision tracking of TrCel7A. Direct observation of motility during degradation reveals processive runs and distinct steps on the scale of 1 nm. Our studies suggest TrCel7A is not mechanically limited, can work against 20 pN loads and speeds up when assisted. Temperature-dependent kinetic studies establish the energy requirements for the fundamental stepping cycle, which likely includes energy from glycosidic bonds and other sources. Through SM measurements of isolated TrCel7A domains, we determine that the catalytic domain alone is sufficient for processive motion, providing insight into TrCel7A's molecular motility mechanism.
authors.affiliation: Department of Chemical and Biomolecular Engineering, Vanderbilt University, PMB 351604, 2301 Vanderbilt Place, Nashville, Tennessee 37235-1604, USA.
authors.forename: Sonia K
authors.initials: SK
authors.lastname: Brady
authors.affiliation: SMART- BioSystems and Micromechanics, National University of Singapore, 1 Create Way, #04-13/14 Enterprise Wing and #B-101, Singapore 138602, Singapore.
authors.forename: Sarangapani
authors.initials: S
authors.lastname: Sreelatha
authors.affiliation: Department of Chemical and Biomolecular Engineering, Vanderbilt University, PMB 351604, 2301 Vanderbilt Place, Nashville, Tennessee 37235-1604, USA.
authors.forename: Yinnian
authors.initials: Y
authors.lastname: Feng
authors.affiliation: Department of Chemical and Biochemical Engineering, Rutgers, State University of New Jersey, 98 Brett Road, Room C-150A, Piscataway, New Jersey 08854, USA.
authors.forename: Shishir P S
authors.initials: SP
authors.lastname: Chundawat
authors.affiliation: Department of Chemical and Biomolecular Engineering, Vanderbilt University, PMB 351604, 2301 Vanderbilt Place, Nashville, Tennessee 37235-1604, USA.|SMART- BioSystems and Micromechanics, National University of Singapore, 1 Create Way, #04-13/14 Enterprise Wing and #B-101, Singapore 138602, Singapore.|Department of Molecular Physiology and Biophysics, Vanderbilt University, PMB 351604, 2301 Vanderbilt Place, Nashville, Tennessee 37235-1604, USA.
authors.forename: Matthew J
authors.initials: MJ
authors.lastname: Lang
mesh_terms: D002482:Cellulose / Q000737:chemistry / Q000378:metabolism*; D043366:Cellulose 1,4-beta-Cellobiosidase / Q000235:genetics / Q000378:metabolism*; D000460:Chlorophyta / Q000737:chemistry; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology; D015966:Gene Expression Regulation, Fungal / Q000502:physiology; D014242:Trichoderma / Q000201:enzymology*
chemical_list: D002482:Cellulose; D043366:Cellulose 1,4-beta-Cellobiosidase