Interfacial molecular interactions of cellobiohydrolase Cel7A and its variants on cellulose

Akshata R Mudinoor; Peter M Goodwin; Raghavendra U Rao; Nardrapee Karuna; Alex Hitomi; Jennifer Nill; Tina Jeoh

doi:10.1186/s13068-020-1649-7

Interfacial molecular interactions of cellobiohydrolase Cel7A and its variants on cellulose

Biotechnol Biofuels. 2020 Jan 18:13:10. doi: 10.1186/s13068-020-1649-7. eCollection 2020.

Authors

Akshata R Mudinoor¹, Peter M Goodwin², Raghavendra U Rao³, Nardrapee Karuna^{1

4}, Alex Hitomi¹, Jennifer Nill^{5

6}, Tina Jeoh¹

Affiliations

¹ 1Biological and Agricultural Engineering, University of California, Davis, One Shields Ave., Davis, CA 95616 USA.
² 2Center for Integrated Nanotechnologies, Los Alamos National Laboratory, Los Alamos, NM 87545 USA.
³ Gracenote, Inc., 2000 Powell Street, Suite 1500, Emeryville, CA 94608 USA.
⁴ 4Biotechnology, Faculty of Engineering and Industrial Technology, Silpakorn University, Nakhon Pathom, 73000 Thailand.
⁵ 5Chemical Engineering, University of California, Davis, One Shields Ave., Davis, CA 95616 USA.
⁶ 6Molecular Biophysics and Integrated Bioimaging, Lawrence Berkeley National Lab, 1 Cyclotron Road, Berkeley, CA 94720 USA.

Abstract

Background: Molecular-scale mechanisms of the enzymatic breakdown of cellulosic biomass into fermentable sugars are still poorly understood, with a need for independent measurements of enzyme kinetic parameters. We measured binding times of cellobiohydrolase Trichoderma reesei Cel7A (Cel7A) on celluloses using wild-type Cel7A (WT_intact), the catalytically deficient mutant Cel7A E212Q (E212Q_intact) and their proteolytically isolated catalytic domains (CD) (WT_core and E212Q_core, respectively). The binding time distributions were obtained from time-resolved, super-resolution images of fluorescently labeled enzymes on cellulose obtained with total internal reflection fluorescence microscopy.

Results: Binding of WT_intact and E212Q_intact on the recalcitrant algal cellulose (AC) showed two bound populations: ~ 85% bound with shorter residence times of < 15 s while ~ 15% were effectively immobilized. The similarity between binding times of the WT and E212Q suggests that the single point mutation in the enzyme active site does not affect the thermodynamics of binding of this enzyme. The isolated catalytic domains, WT_core and E212Q_core, exhibited three binding populations on AC: ~ 75% bound with short residence times of ~ 15 s (similar to the intact enzymes), ~ 20% bound for < 100 s and ~ 5% that were effectively immobilized.

Conclusions: Cel7A binding to cellulose is driven by the interactions between the catalytic domain and cellulose. The cellulose-binding module (CBM) and linker increase the affinity of Cel7A to cellulose likely by facilitating recognition and complexation at the substrate interface. The increased affinity of Cel7A to cellulose by the CBM and linker comes at the cost of increasing the population of immobilized enzyme on cellulose. The residence time (or inversely the dissociation rates) of Cel7A on cellulose is not catalysis limited.

Keywords: Binding lifetime; Catalytic domain; Dissociation rate; Heterogeneous interfacial enzyme kinetics; Single-molecule imaging; Super-resolution; Trichoderma reesei Cel7A.