A Novel Thermophilic Pectate Lyase Containing Two Catalytic Modules of Clostridium stercorarium

The Clostridium stercorarium F-9 pel9A gene encodes a pectate lyase Pel9A consisting of 1,240 amino acids with a molecular weight of 135,171. The mature form of Pel9A is a modular enzyme composed of two family-9 catalytic modules of polysaccharide lyases, CM9-1 and CM9-2, in order from the N terminus. Pel9A showed an overall sequence similarity to the hypothetical pectate lyase PelX of Bacillus halodurans (sequence identity 53%), and CM9-2 showed moderate sequence similarities to some pectate lyases of family 9. Sequence identity between CM9-1 and CM9-2 was 21.3%. The full-length Pel9A lacking the N-terminal signal peptide was expressed, purified, and characterized. The enzyme required Ca2+ ion for its enzyme activity and showed high activity toward polygalacturonic acid but lower activity toward pectin, indicating that Pel9A is a pectate lyase. Immunological analysis using an antiserum raised against the purified enzyme indicated that Pel9A is constitutively synthesized by C. stercorarium F-9.

[1]  M. I. Khan,et al.  Low molecular mass pectate lyase from Fusarium moniliforme: similar modes of chemical and thermal denaturation. , 2004, Biochemical and biophysical research communications.

[2]  N. Keen,et al.  The Role of Pectic Enzymes in Plant Pathogenesis , 1986 .

[3]  G. Davies,et al.  Pectate lyase 10A from Pseudomonas cellulosa is a modular enzyme containing a family 2a carbohydrate-binding module. , 2001, The Biochemical journal.

[4]  M E Watson,et al.  Compilation of published signal sequences. , 1984, Nucleic acids research.

[5]  R. Doi,et al.  Pectate lyase A, an enzymatic subunit of the Clostridium cellulovorans cellulosome , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[6]  W. Schwarz,et al.  Sequence analysis of the Clostridium stercorarium celZ gene encoding a thermoactive cellulase (Avicelase I): Identification of catalytic and cellulose-binding domains , 1990, Molecular and General Genetics MGG.

[7]  K. Sakka,et al.  Nucleotide sequence of the Clostridium stercorarium xynA gene encoding xylanase A: identification of catalytic and cellulose binding domains. , 1993, Bioscience, biotechnology, and biochemistry.

[8]  H. Adelsberger,et al.  Xylan degradation by the thermophile Clostridium stercorarium: Cloning and expression of xylanase, ß-D-xylosidase, and α-L-arabinofuranosidase genes in Escherichia coli , 1990 .

[9]  Pedro M. Coutinho,et al.  Carbohydrate-active enzymes : an integrated database approach , 1999 .

[10]  J. Shine,et al.  Determinant of cistron specificity in bacterial ribosomes , 1975, Nature.

[11]  C. Nagel,et al.  A New Pectic Acid Transeliminase Produced Exocellularly by a Bacillus , 1966 .

[12]  D. Rees,et al.  Structure, conformation, and mechanism in the formation of polysaccharide gels and networks. , 1969, Advances in carbohydrate chemistry and biochemistry.

[13]  K. Sakka,et al.  Nucleotide Sequence of the Clostridium stercorarium xylA Gene Encoding a Bifunctional Protein with β-D-Xylosidase and α-L-Arabinofuranosidase Activities, and Properties of the Translated Product. , 1993, Bioscience, biotechnology, and biochemistry.

[14]  T. Nakaniwa,et al.  Molecular Cloning, DNA Sequence, and Expression of the Gene Encoding for Thermostable Pectate Lyase of Thermophilic Bacillus sp. TS 47 , 2001, Bioscience, biotechnology, and biochemistry.

[15]  Ian K Toth,et al.  Soft rot erwiniae: from genes to genomes. , 2003, Molecular plant pathology.

[16]  K. Sakka,et al.  The multidomain xylanase Xyn10B as a cellulose-binding protein in Clostridium stercorarium. , 2001, FEMS microbiology letters.

[17]  S. Yamamoto,et al.  Molecular cloning and expression of endo-beta-N-acetylglucosaminidase D, which acts on the core structure of complex type asparagine-linked oligosaccharides. , 2001, Journal of biochemistry.

[18]  K. Sakka,et al.  Clostridium thermocellum cellulase CelT, a family 9 endoglucanase without an Ig-like domain or family 3c carbohydrate-binding module , 2002, Applied Microbiology and Biotechnology.

[19]  A. Kondo,et al.  Efficient secretory overexpression of Bacillus subtilis pectate lyase in Escherichia coli and single-step purification , 2002 .

[20]  K. Sakka,et al.  Essential role of the family‐22 carbohydrate‐binding modules for β‐1,3‐1,4‐glucanase activity of Clostridium stercorarium Xyn10B , 2004, FEBS letters.

[21]  U. K. Laemmli,et al.  Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4 , 1970, Nature.

[22]  K. Sakka,et al.  Nucleotide sequence of the Clostridium stercorarium xynB gene encoding an extremely thermostable xylanase, and characterization of the translated product. , 1995, Bioscience, biotechnology, and biochemistry.

[23]  K. Sakka,et al.  Cloning, sequencing, and expression of the gene encoding the Clostridium stercorarium xylanase C in Escherichia coli. , 1999, Bioscience, biotechnology, and biochemistry.

[24]  V. Zverlov,et al.  The thermostable α‐l‐rhamnosidase RamA of Clostridium stercorarium: biochemical characterization and primary structure of a bacterial α‐l‐rhamnoside hydrolase, a new type of inverting glycoside hydrolase , 2000, Molecular microbiology.

[25]  W. D. de Vos,et al.  Molecular and biochemical characterization of the thermoactive family 1 pectate lyase from the hyperthermophilic bacterium Thermotoga maritima. , 2003, The Biochemical journal.

[26]  S. Gold,et al.  Molecular cloning of the structural gene for exopolygalacturonate lyase from Erwinia chrysanthemi EC16 and characterization of the enzyme product , 1990, Journal of bacteriology.

[27]  D Court,et al.  Regulatory sequences involved in the promotion and termination of RNA transcription. , 1979, Annual review of genetics.

[28]  K. Sakka,et al.  Sequencing and Expression of the Gene Encoding the Clostridium stercorarium β-Xylosidase Xyl43B in Escherichia coli , 2004, Bioscience, biotechnology, and biochemistry.

[29]  F. Kittur,et al.  Fusion of family 2b carbohydrate‐binding module increases the catalytic activity of a xylanase from Thermotoga maritima to soluble xylan , 2003, FEBS letters.

[30]  Tohru Kobayashi,et al.  Enzymatic properties of the highly thermophilic and alkaline pectate lyase Pel-4B from alkaliphilic Bacillus sp. strain P-4-N and the entire nucleotide and amino acid sequences , 2001, Extremophiles.

[31]  K. Bronnenmeier,et al.  Purification and properties of a novel type of exo-1,4-beta-glucanase (avicelase II) from the cellulolytic thermophile Clostridium stercorarium. , 1991, European journal of biochemistry.