A new group of exo-acting family 28 glycoside hydrolases of Aspergillus niger that are involved in pectin degradation.

The fungus Aspergillus niger is an industrial producer of pectin-degrading enzymes. The recent solving of the genomic sequence of A. niger allowed an inventory of the entire genome of the fungus for potential carbohydrate-degrading enzymes. By applying bioinformatics tools, 12 new genes, putatively encoding family 28 glycoside hydrolases, were identified. Seven of the newly discovered genes form a new gene group, which we show to encode exoacting pectinolytic glycoside hydrolases. This group includes four exo-polygalacturonan hydrolases (PGAX, PGXA, PGXB and PGXC) and three putative exo-rhamnogalacturonan hydrolases (RGXA, RGXB and RGXC). Biochemical identification using polygalacturonic acid and xylogalacturonan as substrates demonstrated that indeed PGXB and PGXC act as exo-polygalacturonases, whereas PGXA acts as an exo-xylogalacturonan hydrolase. The expression levels of all 21 genes were assessed by microarray analysis. The results from the present study demonstrate that exo-acting glycoside hydrolases play a prominent role in pectin degradation.

[1]  K. D. Macdonald,et al.  The genetics of Aspergillus nidulans. , 1953, Advances in genetics.

[2]  M. Santer,et al.  THE THIOBACILLI, 12 , 1957 .

[3]  Jai-Yun Lim,et al.  Purification and Some Properties of Exo-polygalacturonase from Aspergillus niger Cultured in the Medium Containing Satsuma Mandarin Peel , 1983 .

[4]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[5]  A. Voragen,et al.  Structural features of hairy regions of pectins isolated from apple juice produced by the liquefaction process , 1990 .

[6]  N. Carpita,et al.  Structural models of primary cell walls in flowering plants: consistency of molecular structure with the physical properties of the walls during growth. , 1993, The Plant journal : for cell and molecular biology.

[7]  A. Voragen,et al.  Rhamnogalacturonan [alpha]-L-Rhamnopyranohydrolase (A Novel Enzyme Specific for the Terminal Nonreducing Rhamnosyl Unit in Rhamnogalacturonan Regions of Pectin) , 1994, Plant physiology.

[8]  S. Kauppinen,et al.  Cloning and characterization of two structurally and functionally divergent rhamnogalacturonases from Aspergillus aculeatus. , 1994, The Journal of biological chemistry.

[9]  P. Azadi,et al.  The backbone of the pectic polysaccharide rhamnogalacturonan I is cleaved by an endohydrolase and an endolyase. , 1995, Glycobiology.

[10]  A. Voragen,et al.  Complex Pectins: Structure elucidation using enzymes , 1996 .

[11]  J. Visser,et al.  Primary structure and characterization of an exopolygalacturonase from Aspergillus tubingensis. , 1996, European journal of biochemistry.

[12]  S. Kauppinen,et al.  The crystal structure of rhamnogalacturonase A from Aspergillus aculeatus: a right-handed parallel beta helix. , 1997, Structure.

[13]  J. Visser,et al.  Cloning and characterization of two rhamnogalacturonan hydrolase genes from Aspergillus niger , 1997, Applied and environmental microbiology.

[14]  B Henrissat,et al.  Structural and sequence-based classification of glycoside hydrolases. , 1997, Current opinion in structural biology.

[15]  Mutter,et al.  Rhamnogalacturonan alpha-d-galactopyranosyluronohydrolase. An enzyme that specifically removes the terminal nonreducing galacturonosyl residue in rhamnogalacturonan regions of pectin , 1998, Plant physiology.

[16]  J. Benen,et al.  pgaE encodes a fourth member of the endopolygalacturonase gene family from Aspergillus niger. , 1998, European journal of biochemistry.

[17]  Sean R. Eddy,et al.  Biological Sequence Analysis: Probabilistic Models of Proteins and Nucleic Acids , 1998 .

[18]  H. Schols,et al.  An Enzyme That Specifically Removes the Terminal Nonreducing Galacturonosyl Residue in Rhamnogalacturonan Regions of Pectin 1 , 1998 .

[19]  B. Jones Made by Genetic Engineering , 1999 .

[20]  A. Bairoch,et al.  The SWISS-PROT protein sequence data bank and its supplement TrEMBL in 1999 , 1999, Nucleic Acids Res..

[21]  J. Benen,et al.  Kinetic characterization of Aspergillus niger N400 endopolygalacturonases I, II and C. , 2001, European journal of biochemistry.

[22]  J. Benen,et al.  The exopolygalacturonase from Aspergillus tubingensis is also active on xylogalacturonan , 1999, Biotechnology and applied biochemistry.

[23]  J. Benen,et al.  1.68-Å Crystal Structure of Endopolygalacturonase II fromAspergillus niger and Identification of Active Site Residues by Site-directed Mutagenesis* , 1999, The Journal of Biological Chemistry.

[24]  J. Yates,et al.  Direct analysis of protein complexes using mass spectrometry , 1999, Nature Biotechnology.

[25]  J. Benen,et al.  The Active Site Topology of Aspergillus nigerEndopolygalacturonase II as Studied by Site-directed Mutagenesis* , 2000, The Journal of Biological Chemistry.

[26]  J. Benen,et al.  pgaA and pgaB encode two constitutively expressed endopolygalacturonases of Aspergillus niger. , 2000, The Biochemical journal.

[27]  A. Voragen,et al.  Endo-Xylogalacturonan Hydrolase, a Novel Pectinolytic Enzyme , 2000, Applied and Environmental Microbiology.

[28]  D. Higgins,et al.  T-Coffee: A novel method for fast and accurate multiple sequence alignment. , 2000, Journal of molecular biology.

[29]  J. Benen,et al.  Subsite Mapping of Aspergillus nigerEndopolygalacturonase II by Site-directed Mutagenesis* , 2000, The Journal of Biological Chemistry.

[30]  L. Pařenicová Pectinases of Aspergillus niger: A Molecular and Biochemical Characterisation , 2000 .

[31]  J. Benen,et al.  Characterization of a novel endopolygalacturonase from Aspergillus niger with unique kinetic properties , 2000, FEBS letters.

[32]  W. Shin,et al.  The X-ray structure of Aspergillus aculeatus polygalacturonase and a modeled structure of the polygalacturonase-octagalacturonate complex. , 2001, Journal of molecular biology.

[33]  J. Visser,et al.  Aspergillus Enzymes Involved in Degradation of Plant Cell Wall Polysaccharides , 2001, Microbiology and Molecular Biology Reviews.

[34]  Changing a Single Amino Acid Residue Switches Processive and Non-processive Behavior of Aspergillus nigerEndopolygalacturonase I and II* , 2001, The Journal of Biological Chemistry.

[35]  E. Bonnin,et al.  Purification and characterisation of two exo-polygalacturonases from Aspergillus niger able to degrade xylogalacturonan and acetylated homogalacturonan. , 2002, Biochimica et biophysica acta.

[36]  R. Visser,et al.  Pectin — the Hairy Thing , 2003 .

[37]  J. Vincken,et al.  Degradation of Differently Substituted Xylogalacturonans by Endoxylogalacturonan Hydrolse and Endopolygalacturonases , 2003 .

[38]  Mark S Friedrichs,et al.  Changes in the protein expression of yeast as a function of carbon source. , 2003, Journal of proteome research.

[39]  J. Benen,et al.  Structural insights into the processivity of endopolygalacturonase I from Aspergillus niger , 2003, FEBS letters.

[40]  R. Durbin,et al.  GeneWise and Genomewise. , 2004, Genome research.

[41]  Sudhir Kumar,et al.  MEGA3: Integrated software for Molecular Evolutionary Genetics Analysis and sequence alignment , 2004, Briefings Bioinform..

[42]  J. Visser,et al.  Structure of the Aspergillus niger pelA gene and its expression in Aspergillus niger and Aspergillus nidulans , 1991, Current Genetics.

[43]  J. Visser,et al.  Isolation and transformation of the pyruvate kinase gene of Aspergillus nidulans , 1988, Current Genetics.

[44]  T. Goosen,et al.  Transformation of Aspergillus niger using the homologous orotidine-5′-phosphate-decarboxylase gene , 2004, Current Genetics.

[45]  A. Voragen,et al.  An exogalacturonase from Aspergillus aculeatus able to degrade xylogalacturonan , 1996, Biotechnology Letters.

[46]  A. Voragen,et al.  Mode of action of xylogalacturonan hydrolase towards xylogalacturonan and xylogalacturonan oligosaccharides. , 2005, The Biochemical journal.

[47]  Manesh Shah,et al.  Evaluation of "shotgun" proteomics for identification of biological threat agents in complex environmental matrixes: experimental simulations. , 2005, Analytical chemistry.

[48]  A. Voragen,et al.  Populations having different GalA blocks characteristics are present in commercial pectins which are chemically similar but have different functionalities , 2005 .

[49]  S. Guillotin Studies on the intra- and intermolecular distributions of substituents in commercial pectins , 2005 .

[50]  A. Voragen,et al.  Enzymatic degradation studies of xylogalacturonans from apple and potato, using xylogalacturonan hydrolase , 2006 .