Three-dimensional structure of a thermostable native cellobiohydrolase, CBH IB, and molecular characterization of the cel7 gene from the filamentous fungus, Talaromyces emersonii.

The X-ray structure of native cellobiohydrolase IB (CBH IB) from the filamentous fungus Talaromyces emersonii, PDB 1Q9H, was solved to 2.4 A by molecular replacement. 1Q9H is a glycoprotein that consists of a large, single domain with dimensions of approximately 60 A x 40 A x 50 A and an overall beta-sandwich structure, the characteristic fold of Family 7 glycosyl hydrolases (GH7). It is the first structure of a native glycoprotein and cellulase from this thermophilic eukaryote. The long cellulose-binding tunnel seen in GH7 Cel7A from Trichoderma reesei is conserved in 1Q9H, as are the catalytic residues. As a result of deletions and other changes in loop regions, the binding and catalytic properties of T. emersonii 1Q9H are different. The gene (cel7) encoding CBH IB was isolated from T. emersonii and expressed heterologously with an N-terminal polyHis-tag, in Escherichia coli. The deduced amino acid sequence of cel7 is homologous to fungal cellobiohydrolases in GH7. The recombinant cellobiohydrolase was virtually inactive against methylumberiferyl-cellobioside and chloronitrophenyl-lactoside, but partial activity could be restored after refolding of the urea-denatured enzyme. Profiles of cel7 expression in T. emersonii, investigated by Northern blot analysis, revealed that expression is regulated at the transcriptional level. Putative regulatory element consensus sequences for cellulase transcription factors have been identified in the upstream region of the cel7 genomic sequence.

[1]  J. Kelly,et al.  Specific binding sites in the alcR and alcA promoters of the ethanol regulon for the CREA repressor mediating carbon cataboiite repression in Aspergillus nidulans , 1993, Molecular microbiology.

[2]  S. Withers,et al.  Mechanisms of enzymatic glycoside hydrolysis. , 1994, Current opinion in structural biology.

[3]  E. Bayer,et al.  Isolation and properties of a major cellobiohydrolase from the cellulosome of Clostridium thermocellum , 1991, Journal of bacteriology.

[4]  C. Weigel,et al.  Cloning, sequencing, and heterologous expression of a cellulase-encoding cDNA (cbhl) from Penicillium janthinellum , 1993 .

[5]  K. Myambo,et al.  Molecular Cloning of Exo–Cellobiohydrolase I Derived from Trichoderma Reesei Strain L27 , 1983, Bio/Technology.

[6]  Collaborative Computational,et al.  The CCP4 suite: programs for protein crystallography. , 1994, Acta crystallographica. Section D, Biological crystallography.

[7]  J. Sumitani,et al.  Expression of Aspergillus aculeatus No. F-50 cellobiohydrolase I (cbhI) and beta-glucosidase 1 (bgl1) genes by Saccharomyces cerevisiae. , 1998, Bioscience, biotechnology, and biochemistry.

[8]  H. van Tilbeurgh,et al.  Limited proteolysis of the cellobiohydrolase I from Trichoderma reesei , 1986 .

[9]  D S Moss,et al.  Main-chain bond lengths and bond angles in protein structures. , 1993, Journal of molecular biology.

[10]  H. Masaki,et al.  Isolation of the gene and characterization of the enzymatic properties of a major exoglucanase of humicola grisea without a cellulose-binding domain. , 1998, Journal of Biochemistry (Tokyo).

[11]  M. Penttilä,et al.  Regulation of cellulase gene expression in the filamentous fungus Trichoderma reesei , 1997, Applied and environmental microbiology.

[12]  G. Murshudov,et al.  Refinement of macromolecular structures by the maximum-likelihood method. , 1997, Acta crystallographica. Section D, Biological crystallography.

[13]  S L Mowbray,et al.  Family 7 cellobiohydrolases from Phanerochaete chrysosporium: crystal structure of the catalytic module of Cel7D (CBH58) at 1.32 A resolution and homology models of the isozymes. , 2001, Journal of molecular biology.

[14]  B. Henrissat,et al.  Structures and mechanisms of glycosyl hydrolases. , 1995, Structure.

[15]  M. Penttilä,et al.  The glucose repressor genecre1 ofTrichoderma: Isolation and expression of a full-length and a truncated mutant form , 1996, Molecular and General Genetics MGG.

[16]  M. Coughlan,et al.  The saccharifying ability of the cellulase complex of Talaromyces emersonii and comparison with that of other fungal species. , 1979, The International journal of biochemistry.

[17]  D G Higgins,et al.  CLUSTAL V: multiple alignment of DNA and protein sequences. , 1994, Methods in molecular biology.

[18]  J Navaza,et al.  Implementation of molecular replacement in AMoRe. , 2001, Acta crystallographica. Section D, Biological crystallography.

[19]  P. Considine,et al.  Cellulose hydrolysis by the cellulases produced by Talaromyces emersonii when grown on different inducing substrates. , 1983, Biotechnology and bioengineering.

[20]  G. Davies,et al.  Structure of the endoglucanase I from Fusarium oxysporum: native, cellobiose, and 3,4-epoxybutyl beta-D-cellobioside-inhibited forms, at 2.3 A resolution. , 1997, Biochemistry.

[21]  I. Silva-Pereira,et al.  Substrate-dependent differential expression ofHumicola griseavar.thermoideacellobiohydrolase genes , 2000 .

[22]  M. Harris,et al.  Engineering the exo-loop of Trichoderma reesei cellobiohydrolase, Cel7A. A comparison with Phanerochaete chrysosporium Cel7D. , 2003, Journal of molecular biology.

[23]  B. Henrissat,et al.  Domains in microbial beta-1, 4-glycanases: sequence conservation, function, and enzyme families. , 1991, Microbiological reviews.

[24]  M. Tuohy,et al.  Crystallization and preliminary crystallographic analysis of the catalytic domain cellobiohydrolase I from Talaromyces emersonii. , 2003, Acta crystallographica. Section D, Biological crystallography.

[25]  J. V. van Wyk,et al.  Biodegradation of wastepaper by cellulase from Trichoderma viride. , 2003, Bioresource technology.

[26]  Marco M. C. Gielkens,et al.  Two Cellobiohydrolase-Encoding Genes from Aspergillus niger Require d-Xylose and the Xylanolytic Transcriptional Activator XlnR for Their Expression , 1999, Applied and Environmental Microbiology.

[27]  M. Claeyssens,et al.  A hydrophobic platform as a mechanistically relevant transition state stabilising factor appears to be present in the active centre of all glycoside hydrolases , 2003, FEBS letters.

[28]  H. Ronne,et al.  Yeast MIG1 repressor is related to the mammalian early growth response and Wilms' tumour finger proteins. , 1990, The EMBO journal.

[29]  L. Fägerstam,et al.  The primary structure of a 1,4‐β‐glucan cellobiohydrolase from the fungus Trichoderma reesei QM 9414 , 1984 .

[30]  T. A. Jones,et al.  High-resolution crystal structures reveal how a cellulose chain is bound in the 50 A long tunnel of cellobiohydrolase I from Trichoderma reesei. , 1998, Journal of molecular biology.

[31]  T. Littlejohn,et al.  Analysis of the site of action of the amdR product for regulation of the amdS gene of Aspergillus nidulans , 1992, Molecular and General Genetics MGG.

[32]  Tetsuo Kobayashi,et al.  Structure and expression properties of the endo-β-1,4-glucanase A gene from the filamentous fungus Aspergillus nidulans , 1999 .

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

[34]  M. Coughlan,et al.  Sorption of Talaromyces emersonii cellulase on cellulosic substrates , 1983, Biotechnology and bioengineering.

[35]  P. Broda,et al.  Rapid preparation of DNA from filamentous fungi , 1985 .

[36]  M. D. Joshi,et al.  Interaction of soluble cellooligosaccharides with the N-terminal cellulose-binding domain of Cellulomonas fimi CenC 2. NMR and ultraviolet absorption spectroscopy. , 1996, Biochemistry.

[37]  Thomas L. Madden,et al.  Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. , 1997, Nucleic acids research.

[38]  U. Heinemann,et al.  Crystal Structure and Site-directed Mutagenesis of Bacillus macerans Endo-1,31,4--glucanase (*) , 1995, The Journal of Biological Chemistry.

[39]  A. McHale,et al.  PURIFICATION OF BETA-D-GLUCOSIDE GLUCOHYDROLASES OF TALAROMYCES-EMERSONII , 1988 .

[40]  L. Walker,et al.  Enzymatic hydrolysis of cellulose: An overview , 1991 .

[41]  M. Penttilä,et al.  Isolation of the ace1 Gene Encoding a Cys2-His2 Transcription Factor Involved in Regulation of Activity of the Cellulase Promoter cbh1of Trichoderma reesei * , 2000, The Journal of Biological Chemistry.

[42]  P. Birch,et al.  Isolation, characterization, and analysis of the expression of the cbhII gene of Phanerochaete chrysosporium , 1994, Applied and environmental microbiology.

[43]  M. Penttilä,et al.  Role of the interdomain linker peptide of Trichoderma reesei cellobiohydrolase I in its interaction with crystalline cellulose. , 1993, The Journal of biological chemistry.

[44]  G J Kleywegt,et al.  The crystal structure of the catalytic core domain of endoglucanase I from Trichoderma reesei at 3.6 A resolution, and a comparison with related enzymes. , 1997, Journal of molecular biology.

[45]  J. Vandekerckhove,et al.  Studies of the cellulolytic system of Trichoderma reesei QM 9414. Analysis of domain function in two cellobiohydrolases by limited proteolysis. , 1988, European journal of biochemistry.

[46]  V S Lamzin,et al.  Automated refinement of protein models. , 1993, Acta crystallographica. Section D, Biological crystallography.

[47]  S. Withers,et al.  Approaches to labeling and identification of active site residues in glycosidases , 1995, Protein science : a publication of the Protein Society.

[48]  H. Lawford,et al.  Cellulosic fuel ethanol , 2003, Applied biochemistry and biotechnology.

[49]  N. Panasik,et al.  Distributions of structural features contributing to thermostability in mesophilic and thermophilic α/β barrel glycosyl hydrolases , 2000 .

[50]  M. Penttilä,et al.  ACEI of Trichoderma reesei Is a Repressor of Cellulase and Xylanase Expression , 2003, Applied and Environmental Microbiology.

[51]  S. Covert,et al.  Identification of the gene encoding the major cellobiohydrolase of the white rot fungus Phanerochaete chrysosporium , 1993, Applied and environmental microbiology.

[52]  G. Pettersson,et al.  Progress curves--a mean for functional classification of cellulases. , 1998, European journal of biochemistry.

[53]  S. McKnight,et al.  Eukaryotic transcriptional regulatory proteins. , 1989, Annual review of biochemistry.

[54]  S. Zeilinger,et al.  Crel, the carbon catabolite repressor protein from Trichoderma reesei , 1995, FEBS letters.

[55]  H. Masaki,et al.  Molecular cloning and expression of the novel fungal beta-glucosidase genes from Humicola grisea and Trichoderma reesei. , 1999, Journal of biochemistry.

[56]  I. Silva-Pereira,et al.  Substrate-dependent differential expression of Humicola grisea var. thermoidea cellobiohydrolase genes. , 2000, Canadian journal of microbiology.

[57]  C. Collins,et al.  Molecular cloning, transcriptional, and expression analysis of the first cellulase gene (cbh2), encoding cellobiohydrolase II, from the moderately thermophilic fungus Talaromyces emersonii and structure prediction of the gene product. , 2003, Biochemical and biophysical research communications.

[58]  Marc Claeyssens,et al.  Kinetic parameters and mode of action of the cellobiohydrolases produced by Talaromyces emersonii. , 2002, Biochimica et biophysica acta.

[59]  S. Withers,et al.  Direct 1H n.m.r. determination of the stereochemical course of hydrolyses catalysed by glucanase components of the cellulase complex. , 1986, Biochemical and biophysical research communications.

[60]  S. Covert,et al.  Genomic organization of a cellulase gene family in Phanerochaete chrysosporium , 1992, Current Genetics.

[61]  T. Wood,et al.  Isolation and characterization of the 1,4-beta-D-glucan glucanohydrolases of Talaromyces emersonii. , 1985, The Biochemical journal.

[62]  Michael P. Coughlan,et al.  The Properties of Fungal and Bacterial Cellulases with Comment on their Production and Application , 1985 .

[63]  M. Penttilä,et al.  The glucose repressor gene , 1996 .