Studies of Thermobifida fusca plant cell wall degrading enzymes.

I have been studying the Thermobifida fusca cellulose degrading proteins for the past 25 years. In this period, we have purified and characterized the six extracellular cellulases and an intracellular beta- glucosidase used by T. fusca for cellulose degradation, cloned and sequenced the structural genes encoding these enzymes, and helped to determine the 3-dimensional structures of two of the cellulase catalytic domains. This research determined the mechanism of a novel class of cellulase, family 9 processive endoglucanases, and helped to show that there were two types of exocellulases, ones that attacked the non-reducing ends of cellulose and ones that attacked the reducing ends. It also led to the sequencing of the T. fusca genome by the DOE Joint Genome Institute. We have studied the mechanisms that regulate T. fusca cellulases and have shown that cellobiose is the inducer and that cellulase synthesis is repressed by any good carbon source. A regulatory protein (CelR) that functions in the induction control has been purified, characterized, and its structural gene cloned and expressed in E. coli. I have also carried out research on two rumen bacteria, Prevotella ruminicola and Fibrobacter succinogenes, in collaboration with Professor James Russell, helping to arrange for the genomes of these two organisms to be sequenced by TIGR, funded by a USDA grant to the North American Consortium for Genomics of Fibrolytic Ruminal Biology.

[1]  L. Walker,et al.  Activity studies of eight purified cellulases: Specificity, synergism, and binding domain effects , 1993, Biotechnology and bioengineering.

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

[3]  J. Knowles,et al.  Three-dimensional structure of cellobiohydrolase II from Trichoderma reesei. , 1990, Science.

[4]  D. Wilson,et al.  Cloning of a Thermomonospora fusca xylanase gene and its expression in Escherichia coli and Streptomyces lividans , 1989, Journal of bacteriology.

[5]  D. Kilburn,et al.  The tertiary structure of endo-beta-1,4-glucanase B (CenB), a multidomain cellulase from the bacterium Cellulomonas fimi. , 1992, Glycobiology.

[6]  D. Wilson,et al.  Effects of noncatalytic residue mutations on substrate specificity and ligand binding of Thermobifida fusca endocellulase cel6A. , 2000, European journal of biochemistry.

[7]  J. Martín,et al.  Cloning, expression in Streptomyces lividans and biochemical characterization of a thermostable endo-β-1,4-xylanase of Thermomonospora alba UL JB1 with cellulose-binding ability , 1997, Applied Microbiology and Biotechnology.

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

[9]  W. Steiner,et al.  Cellulose hydrolysis by the cellulases from Trichoderma reesei: a new model for synergistic interaction. , 1994, The Biochemical journal.

[10]  D. Wilson,et al.  Cloning and sequencing of a Bacteroides ruminicola B(1)4 endoglucanase gene , 1990, Journal of bacteriology.

[11]  G. Kleywegt,et al.  The active site of cellobiohydrolase Cel6A from Trichoderma reesei: the roles of aspartic acids D221 and D175. , 2002, Journal of the American Chemical Society.

[12]  D. Rawlings,et al.  Autoregulation of the pTF-FC2 Proteic Poison-Antidote Plasmid Addiction System (pas) Is Essential for Plasmid Stabilization , 1998, Journal of bacteriology.

[13]  D. Wilson,et al.  DNA sequences and expression in Streptomyces lividans of an exoglucanase gene and an endoglucanase gene from Thermomonospora fusca , 1993, Applied and environmental microbiology.

[14]  D. Wilson,et al.  Engineering cellulase mixtures by varying the mole fraction of Thermomonospora fusca E5 and E3, Trichoderma reesei CBHI, and Caldocellum saccharolyticum β‐glucosidase , 1993, Biotechnology and bioengineering.

[15]  D. Wilson,et al.  Characterization of a Thermomonospora fusca exocellulase. , 1995, Biochemistry.

[16]  D. Irwin,et al.  Purification and characterization of two β-1,4-endoglucanases from Thermomonospora fusca , 1985 .

[17]  L. Walker,et al.  The impact of initial particle size on the fragmentation of cellulose by the cellulase of thermomonospora fusca , 1991 .

[18]  C. Forsberg,et al.  A Cold-Active Glucanase from the Ruminal BacteriumFibrobacter succinogenes S85 , 1999, Applied and Environmental Microbiology.

[19]  P. Karplus,et al.  Crystal structure of the catalytic domain of a thermophilic endocellulase. , 1993, Biochemistry.

[20]  D. Wilson,et al.  A Bacteroides ruminicola 1,4-beta-D-endoglucanase is encoded in two reading frames , 1991, Journal of bacteriology.

[21]  D. Wilson,et al.  Mechanistic studies of active site mutants of Thermomonospora fusca endocellulase E2. , 1999, Biochemistry.

[22]  M. Bothwell,et al.  Binding capacities for Thermomonospora fusca E3, E4 and E5, the E3 binding domain, and Trichoderma reesei CBHI on Avicel and bacterial microcrystalline cellulose , 1997 .

[23]  A. Darke,et al.  In vitro assembly of cellulose/xyloglucan networks: ultrastructural and molecular aspects , 1995 .

[24]  D. Wilson,et al.  Transcription of the celE gene in Thermomonospora fusca , 1988, Journal of bacteriology.

[25]  D. Kilburn,et al.  Non–Hydrolytic Disruption of Cellulose Fibres by the Binding Domain of a Bacterial Cellulase , 1991, Bio/Technology.

[26]  O. Griffith,et al.  In vivo studies of cysteine metabolism. Use of D-cysteinesulfinate, a novel cysteinesulfinate decarboxylase inhibitor, to probe taurine and pyruvate synthesis. , 1988, The Journal of biological chemistry.

[27]  Binding reversibility and surface exchange of Thermomonospora fusca E3 and E5 and Trichoderma reesei CBHI , 1997 .

[28]  D. Wilson,et al.  Identification of a celE-binding protein and its potential role in induction of the celE gene in Thermomonospora fusca , 1988, Journal of bacteriology.

[29]  W. Steiner,et al.  Cellulose hydrolysis by the cellulases from Trichoderma reesei: adsorptions of two cellobiohydrolases, two endocellulases and their core proteins on filter paper and their relation to hydrolysis. , 1994, The Biochemical journal.

[30]  D. Wilson,et al.  Cloning of Thermomonospora fusca genes coding for beta 1-4 endoglucanases E1, E2 and E5. , 1988, Gene.

[31]  L. Walker,et al.  Cooperative and Competitive Binding in Synergistic Mixtures of Thermobifida fuscaCellulases Cel5A, Cel6B, and Cel9A , 2002, Biotechnology progress.

[32]  D. Wilson,et al.  Characterization and Cloning of CelR, a Transcriptional Regulator of Cellulase Genes from Thermomonospora fusca * , 1999, The Journal of Biological Chemistry.

[33]  A. Mccarthy,et al.  Purification and Cooperative Activity of Enzymes Constituting the Xylan-Degrading System of Thermomonospora fusca , 1991, Applied and environmental microbiology.

[34]  D. Wilson,et al.  Cloning, sequencing, and expression of a Thermomonospora fusca protease gene in Streptomyces lividans , 1996, Applied and environmental microbiology.

[35]  D. Wilson.  Cellulases of Thermomonospora fusca , 1988 .

[36]  D. Wilson,et al.  DNA sequences of three beta-1,4-endoglucanase genes from Thermomonospora fusca , 1991, Journal of bacteriology.

[37]  J. Rose,et al.  Cloning, expression and characterization of a family-74 xyloglucanase from Thermobifida fusca. , 2003, European journal of biochemistry.

[38]  L. Walker,et al.  Measuring fragmentation of cellulose by Thermomonospora fusca cellulase , 1990 .

[39]  M. Lascombe,et al.  The crystal structure of a family 5 endoglucanase mutant in complexed and uncomplexed forms reveals an induced fit activation mechanism. , 1996, Journal of molecular biology.

[40]  A. Collmer,et al.  Cloning and Expression of a Thermomonospora YX Endocellulase Gene in E. coli , 1983, Bio/Technology.

[41]  R. Warren.  Microbial hydrolysis of polysaccharides. , 1996, Annual review of microbiology.

[42]  H. Gilbert,et al.  Gene sequence and properties of CelI, a family E endoglucanase from Clostridium thermocellum. , 1993, Journal of general microbiology.

[43]  D. Wilson,et al.  Surface residue mutations which change the substrate specificity of Thermomonospora fusca endoglucanase E2. , 1997, Journal of biotechnology.

[44]  Computational and experimental studies of the catalytic mechanism of Thermobifida fusca cellulase Cel6A (E2). , 2003, Protein engineering.

[45]  D. Wilson,et al.  Characterization and sequence of a Thermomonospora fusca xylanase , 1994, Applied and environmental microbiology.

[46]  J. Kinsella,et al.  Purification and characterization of the heat-stable serine proteinase from Thermomonospora fusca YX. , 1987, The Biochemical journal.

[47]  M. Penttilä,et al.  EGIII, a new endoglucanase from Trichoderma reesei: the characterization of both gene and enzyme. , 1988, Gene.

[48]  B. Ganem,et al.  Identification of two functionally different classes of exocellulases. , 1996, Biochemistry.

[49]  M. Bothwell,et al.  Synergism between pure Thermomonospora fusca and Trichoderma reesei cellulases , 1993 .

[50]  B. Henrissat,et al.  Structures of oligosaccharide-bound forms of the endoglucanase V from Humicola insolens at 1.9 A resolution. , 1995, Biochemistry.

[51]  D. Wilson,et al.  Regulation of β-1,4-Endoglucanase Synthesis in Thermomonospora fusca , 1987 .

[52]  D. Kilburn,et al.  Cellulose-binding domains : classification and properties , 1995 .

[53]  D. Wilson,et al.  Cloning of the Thermomonospora fusca Endoglucanase E2 Gene in Streptomyces lividans: Affinity Purification and Functional Domains of the Cloned Gene Product , 1988, Applied and environmental microbiology.

[54]  R. Haser,et al.  Crystal Structures of the Cellulase Cel48F in Complex with Inhibitors and Substrates Give Insights Into its Processive Action , 2000 .

[55]  D. Wilson,et al.  A celR Mutation Affecting Transcription of Cellulase Genes in Thermobifida fusca , 2000, Journal of bacteriology.

[56]  D. Wilson,et al.  Substrate heterogeneity causes the nonlinear kinetics of insoluble cellulose hydrolysis. , 1999, Biotechnology and bioengineering.

[57]  D. Wilson,et al.  Properties of a genetically reconstructed Prevotella ruminicola endoglucanase , 1992, Applied and environmental microbiology.

[58]  P. Karplus,et al.  Structure and mechanism of endo/exocellulase E4 from Thermomonospora fusca , 1997, Nature Structural Biology.

[59]  Shenmin Zhang,et al.  Roles of the Catalytic Domain and Two Cellulose Binding Domains of Thermomonospora fusca E4 in Cellulose Hydrolysis , 1998, Journal of bacteriology.

[60]  D. Kilburn,et al.  Cellobiohydrolase A (CbhA) from the cellulolytic bacterium Cellulomonas fimi is a β‐1,4‐exoceilobiohydrolase analogous to Trichoderma reesei CBH II , 1994, Molecular microbiology.

[61]  L. Walker,et al.  Binding mechanisms for Thermobifida fusca Cel5A, Cel6B, and Cel48A cellulose‐binding modules on bacterial microcrystalline cellulose , 2002, Biotechnology and bioengineering.

[62]  B. Henrissat,et al.  Imaging the Enzymatic Digestion of Bacterial Cellulose Ribbons Reveals the Endo Character of the Cellobiohydrolase Cel6A from Humicola insolens and Its Mode of Synergy with Cellobiohydrolase Cel7A , 2000, Applied and Environmental Microbiology.

[63]  D. Wilson,et al.  Biochemistry and genetics of actinomycete cellulases. , 1992, Critical reviews in biotechnology.

[64]  D. Wilson,et al.  Cloning and Biochemical Characterization of BglC, a β-Glucosidase from the Cellulolytic Actinomycete Thermobifida fusca , 2001, Current Microbiology.