Aromatic residues surrounding the active site tunnel of TfCel48A influence activity, processivity, and synergistic interactions with other cellulases

Cel48A of Thermobifida fusca (TfCel48A) is a processive exocellulase that contains an active site tunnel and digests lignocellulosic biomass via synergistic interactions between different cellulases. Cel48A possesses a number of aromatic amino acids lining the tunnel entrance, which are highly conserved across a diverse number of microbial species and appear to play a role in the selection and threading of individual strands of cellulose from highly recalcitrant substrates. In this study, we sought to further elucidate the roles of these tunnel entrance aromatic amino acids by creating a series of double mutants and examining their effect on TfCel48A activity, processivity, and synergistic interactions with the well‐studied processive endocellulase TfCel9A. Our results provide further insight concerning the mechanism of Cel48A kinetics with soluble and insoluble substrates and could play an influential role in the application of Cel48A and other exocellulases for industrial purposes.

[1]  A. Ellis,et al.  Increased solubility of plant core pulp cellulose for regenerated hydrogels through electron beam irradiation , 2018, Cellulose.

[2]  Christina M. Payne,et al.  Correlation of structure, function and protein dynamics in GH7 cellobiohydrolases from Trichoderma atroviride, T. reesei and T. harzianum , 2018, Biotechnology for Biofuels.

[3]  M. Himmel,et al.  Natural diversity of glycoside hydrolase family 48 exoglucanases: insights from structure , 2017, Biotechnology for Biofuels.

[4]  T. Furuta,et al.  Alanine substitution in cellobiohydrolase provides new insights into substrate threading , 2017, Scientific Reports.

[5]  N. Karuna,et al.  Mechanistic kinetic models of enzymatic cellulose hydrolysis—A review , 2017, Biotechnology and bioengineering.

[6]  Supratim Datta,et al.  Understanding the role of residues around the active site tunnel towards generating a glucose-tolerant &bgr;-glucosidase from Agrobacterium tumefaciens 5A , 2017, Protein engineering, design & selection : PEDS.

[7]  K. Igarashi,et al.  Crystal structure of a family 6 cellobiohydrolase from the basidiomycete Phanerochaete chrysosporium , 2017, Acta crystallographica. Section F, Structural biology communications.

[8]  V. Eijsink,et al.  Thermodynamics of tunnel formation upon substrate binding in a processive glycoside hydrolase. , 2017, Archives of biochemistry and biophysics.

[9]  C. Wyman,et al.  Dynamic changes of substrate reactivity and enzyme adsorption on partially hydrolyzed cellulose , 2017, Biotechnology and bioengineering.

[10]  N. Karuna,et al.  The productive cellulase binding capacity of cellulosic substrates , 2017, Biotechnology and bioengineering.

[11]  Supratim Datta,et al.  Exploiting non-conserved residues to improve activity and stability of Halothermothrix orenii β-glucosidase , 2017, Applied Microbiology and Biotechnology.

[12]  Johan P. Olsen,et al.  Anomeric Selectivity and Product Profile of a Processive Cellulase. , 2017, Biochemistry.

[13]  P. Westh,et al.  Inter-domain Synergism Is Required for Efficient Feeding of Cellulose Chain into Active Site of Cellobiohydrolase Cel7A* , 2016, The Journal of Biological Chemistry.

[14]  M. Himmel,et al.  Simulation studies of substrate recognition by the exocellulase CelF from Clostridium cellulolyticum , 2016, Biotechnology and bioengineering.

[15]  J. Ottesen,et al.  Rate of Threading a Cellulose Chain into the Binding Tunnel of a Cellulase. , 2016, The journal of physical chemistry. B.

[16]  P. Westh,et al.  Free Energy Diagram for the Heterogeneous Enzymatic Hydrolysis of Glycosidic Bonds in Cellulose* , 2015, The Journal of Biological Chemistry.

[17]  P. Toscas,et al.  C/N Ratio Drives Soil Actinobacterial Cellobiohydrolase Gene Diversity , 2015, Applied and Environmental Microbiology.

[18]  Johan P. Olsen,et al.  Probing Substrate Interactions in the Active Tunnel of a Catalytically Deficient Cellobiohydrolase (Cel7)* , 2014, The Journal of Biological Chemistry.

[19]  Christina M. Payne,et al.  Towards a molecular-level theory of carbohydrate processivity in glycoside hydrolases. , 2014, Current opinion in biotechnology.

[20]  O. V. Oliveira.  Molecular Dynamics and Metadynamics Simulations of the Cellulase Cel48F. , 2014 .

[21]  Osmair Vital de Oliveira.  Molecular Dynamics and Metadynamics Simulations of the Cellulase Cel48F , 2014, Enzyme research.

[22]  Timothy C Schutt,et al.  Computational evaluation of the dynamic fluctuations of peripheral loops enclosing the catalytic tunnel of a family 7 cellobiohydrolase. , 2014, The journal of physical chemistry. B.

[23]  M. Himmel,et al.  Cel48A from Thermobifida fusca: Structure and site directed mutagenesis of key residues , 2014, Biotechnology and bioengineering.

[24]  Qing Yang,et al.  Structural characteristics of an insect group I chitinase, an enzyme indispensable to moulting , 2014, Acta crystallographica. Section D, Biological crystallography.

[25]  D. Wilson,et al.  A Distinct Model of Synergism between a Processive Endocellulase (TfCel9A) and an Exocellulase (TfCel48A) from Thermobifida fusca , 2013, Applied and Environmental Microbiology.

[26]  Maxim Kostylev,et al.  Two-parameter kinetic model based on a time-dependent activity coefficient accurately describes enzymatic cellulose digestion. , 2013, Biochemistry.

[27]  P. Westh,et al.  A steady‐state theory for processive cellulases , 2013, The FEBS journal.

[28]  Christina M. Payne,et al.  Binding site dynamics and aromatic-carbohydrate interactions in processive and non-processive family 7 glycoside hydrolases. , 2013, The journal of physical chemistry. B.

[29]  A. Koivula,et al.  The Tryptophan Residue at the Active Site Tunnel Entrance of Trichoderma reesei Cellobiohydrolase Cel7A Is Important for Initiation of Degradation of Crystalline Cellulose* , 2013, The Journal of Biological Chemistry.

[30]  J. Ståhlberg,et al.  The structure of a bacterial cellobiohydrolase: the catalytic core of the Thermobifida fusca family GH6 cellobiohydrolase Cel6B. , 2013, Journal of molecular biology.

[31]  P. Väljamäe,et al.  Endo-exo Synergism in Cellulose Hydrolysis Revisited* , 2012, The Journal of Biological Chemistry.

[32]  Michael R. Shirts,et al.  Product Binding Varies Dramatically between Processive and Nonprocessive Cellulase Enzymes* , 2012, The Journal of Biological Chemistry.

[33]  D. Wilson,et al.  Synergistic interactions in cellulose hydrolysis , 2012 .

[34]  Clare McCabe,et al.  Multiple Functions of Aromatic-Carbohydrate Interactions in a Processive Cellulase Examined with Molecular Simulation* , 2011, The Journal of Biological Chemistry.

[35]  D. Wilson,et al.  Determination of the Catalytic Base in Family 48 Glycosyl Hydrolases , 2011, Applied and Environmental Microbiology.

[36]  J. Brady,et al.  Glucose interactions with a model peptide , 2011, Proteins.

[37]  J. Moran‐Mirabal,et al.  Reversibility and binding kinetics of Thermobifida fusca cellulases studied through fluorescence recovery after photobleaching microscopy. , 2011, Biophysical chemistry.

[38]  P. Väljamäe,et al.  Processivity of Cellobiohydrolases Is Limited by the Substrate* , 2010, The Journal of Biological Chemistry.

[39]  Ross C Walker,et al.  Computational simulations of the Trichoderma reesei cellobiohydrolase I acting on microcrystalline cellulose Ibeta: the enzyme-substrate complex. , 2009, Carbohydrate research.

[40]  D. Wilson,et al.  Processivity, Synergism, and Substrate Specificity of Thermobifida fusca Cel6B , 2009, Applied and Environmental Microbiology.

[41]  V. Eijsink,et al.  Aromatic Residues in the Catalytic Center of Chitinase A from Serratia marcescens Affect Processivity, Enzyme Activity, and Biomass Converting Efficiency* , 2009, Journal of Biological Chemistry.

[42]  G. Bergstrom,et al.  An optimized microplate assay system for quantitative evaluation of plant cell wall–degrading enzyme activity of fungal culture extracts , 2009, Biotechnology and bioengineering.

[43]  M. Pedraza-Reyes,et al.  Expression, Characterization and Synergistic Interactions of Myxobacter Sp. AL-1 Cel9 and Cel48 Glycosyl Hydrolases , 2008, International journal of molecular sciences.

[44]  B. Synstad,et al.  Costs and benefits of processivity in enzymatic degradation of recalcitrant polysaccharides , 2006, Proceedings of the National Academy of Sciences.

[45]  Shenmin Zhang,et al.  Cloning, expression and characterization of a family 48 exocellulase, Cel48A, from Thermobifida fusca. , 2000, European journal of biochemistry.

[46]  Shenmin Zhang,et al.  Site-directed mutation of noncatalytic residues of Thermobifida fusca exocellulase Cel6B. , 2000, European journal of biochemistry.

[47]  G. Pettersson,et al.  Acid hydrolysis of bacterial cellulose reveals different modes of synergistic action between cellobiohydrolase I and endoglucanase I. , 1999, European Journal of Biochemistry.

[48]  L. Ruohonen,et al.  Tryptophan 272: an essential determinant of crystalline cellulose degradation by Trichoderma reesei cellobiohydrolase Cel6A , 1998, FEBS letters.

[49]  K Schulten,et al.  VMD: visual molecular dynamics. , 1996, Journal of molecular graphics.

[50]  M. Lever.  Carbohydrate determination with 4-hydroxybenzoic acid hydrazide (PAHBAH): effect of bismuth on the reaction. , 1977, Analytical biochemistry.

[51]  D. Wilson,et al.  Characterization of cellulose crystallinity after enzymatic treatment using Fourier transform infrared spectroscopy (FTIR) , 2017, Cellulose.

[52]  V. Eijsink,et al.  Measuring processivity. , 2012, Methods in enzymology.

[53]  D. Wilson,et al.  Cellulase processivity. , 2012, Methods in molecular biology.

[54]  D. Wilson,et al.  Studies of Thermobifida fusca plant cell wall degrading enzymes. , 2004, Chemical record.