Molecular and biochemical characterization of the thermoactive family 1 pectate lyase from the hyperthermophilic bacterium Thermotoga maritima.

The ability of the hyperthermophilic bacterium Thermotoga maritima to grow on pectin as a sole carbon source coincides with the secretion of a pectate lyase A (PelA) in the extracellular medium. The pel A gene of T. maritima was functionally expressed in Escherichia coli as the first heterologously produced thermophilic pectinase, and purified to homogeneity. Gel filtration indicated that the native form of PelA is tetrameric. Highest activity (422 units/mg, with a K(m) of 0.06 mM) was demonstrated on polygalacturonic acid (PGA), whereas pectins with an increasing degree of methylation were degraded at a decreasing rate. In the tradition of pectate lyases, PelA demonstrated full dependency on Ca(2+) for stability and activity. The enzyme is highly thermoactive and thermostable, operating optimally at 90 degrees C and pH 9.0, with a half-life for thermal inactivation of almost 2 h at 95 degrees C, and an apparent melting temperature of 102.5 degrees C. Detailed characterization of the product formation with PGA indicated that PelA has a unique eliminative exo-cleavage pattern liberating unsaturated trigalacturonate as the major product, in contrast with unsaturated digalacturonate for other exopectate lyases known. The unique exo-acting mode of action was supported by progression profiles of PelA on oligogalacturonides (degree of polymerization, 3-8) and the examination of the bond cleavage frequencies.

[1]  Robert Huber,et al.  Thermotoga maritima sp. nov. represents a new genus of unique extremely thermophilic eubacteria growing up to 90°C , 1986, Archives of Microbiology.

[2]  J. Benen,et al.  Characterization of Aspergillus niger pectate lyase A. , 2000, Biochemistry.

[3]  E. Stackebrandt,et al.  Description of Caldicellulosiruptor saccharolyticus gen. nov., sp. nov: an obligately anaerobic, extremely thermophilic, cellulolytic bacterium. , 1994, FEMS microbiology letters.

[4]  G. Heijne.  A new method for predicting signal sequence cleavage sites. , 1986 .

[5]  J. Benen,et al.  Characterization of the Exopolygalacturonate Lyase PelX of Erwinia chrysanthemi 3937 , 1999, Journal of bacteriology.

[6]  A. Voragen.  Characterization of pectin lyases on pectins and methyl oligogalacturonates , 1972 .

[7]  F. A. Rainey,et al.  Purification and characterization of thermostable pectate-lyases from a newly isolated thermophilic bacterium, Thermoanaerobacter italicus sp. nov. , 1997, Extremophiles.

[8]  Elisabeth Helmke,et al.  Cloning of two pectate lyase genes from the marine Antarctic bacterium Pseudoalteromonas haloplanktis strain ANT/505 and characterization of the enzymes , 2001, Extremophiles.

[9]  K. Shockley,et al.  Regulation of Endo-Acting Glycosyl Hydrolases in the Hyperthermophilic Bacterium Thermotoga maritima Grown on Glucan- and Mannan-Based Polysaccharides , 2002, Applied and Environmental Microbiology.

[10]  O. Mayans,et al.  Two crystal structures of pectin lyase A from Aspergillus reveal a pH driven conformational change and striking divergence in the substrate-binding clefts of pectin and pectate lyases. , 1997, Structure.

[11]  O. Zabotina,et al.  Esterification and glycosydation of oligogalacturonides: examination of the reaction products using MALDI-TOF MS and HPAEC , 2000 .

[12]  S. Salzberg,et al.  Evidence for lateral gene transfer between Archaea and Bacteria from genome sequence of Thermotoga maritima , 1999, Nature.

[13]  R. Huber,et al.  The Order Thermotogales , 1992 .

[14]  Peer Bork,et al.  Recent improvements to the SMART domain-based sequence annotation resource , 2002, Nucleic Acids Res..

[15]  E. Vandamme,et al.  Pectin, pectinase and protopectinase: production, properties, and applications. , 1993, Advances in applied microbiology.

[16]  M. Danson,et al.  Assaying activity and assessing thermostability of hyperthermophilic enzymes. , 2001, Methods in enzymology.

[17]  M Takao,et al.  Purification and Characterization of Thermostable Pectate Lyase with Protopectinase Activity from Thermophilic Bacillus sp. TS 47 , 2000, Bioscience, biotechnology, and biochemistry.

[18]  A. Collmer,et al.  Assay methods for pectic enzymes , 1988 .

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

[20]  V. Shevchik,et al.  The Exopolygalacturonate Lyase PelW and the Oligogalacturonate Lyase Ogl, Two Cytoplasmic Enzymes of Pectin Catabolism in Erwinia chrysanthemi 3937 , 1999, Journal of bacteriology.

[21]  Robert K. Scopes,et al.  Protein Purification: Principles and Practice , 1982 .

[22]  J. Wiegel,et al.  Isolation from soil and properties of the extreme thermophile Clostridium thermohydrosulfuricum , 1979, Journal of bacteriology.

[23]  K. Horikoshi,et al.  Analysis of the genome of an alkaliphilic Bacillus strain from an industrial point of view , 2000, Extremophiles.

[24]  R. Pickersgill,et al.  The structure of Bacillus subtilis pectate lyase in complex with calcium , 1994, Nature Structural Biology.

[25]  B. Patel,et al.  Caldicellulosiruptor owensensis sp. nov., an anaerobic, extremely thermophilic, xylanolytic bacterium. , 1998, International journal of systematic bacteriology.

[26]  J. Mikkelsen,et al.  Mode of Action of Pectin Lyase A of Aspergillus nigeron Differently C6-substituted Oligogalacturonides* , 2002, The Journal of Biological Chemistry.

[27]  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.

[28]  B. Ahring,et al.  Caldicellulosiruptor kristjanssonii sp. nov., a cellulolytic, extremely thermophilic, anaerobic bacterium. , 1999, International journal of systematic bacteriology.

[29]  D R Kashyap,et al.  Applications of pectinases in the commercial sector: a review. , 2001, Bioresource technology.

[30]  N. Hugouvieux-Cotte-Pattat,et al.  Comparative analysis of the five major Erwinia chrysanthemi pectate lyases: enzyme characteristics and potential inhibitors , 1997, Journal of bacteriology.

[31]  L. Lo Leggio,et al.  Crystallization and preliminary X-ray characterization of a thermostable pectate lyase from Thermotoga maritima. , 2002, Acta crystallographica. Section D, Biological crystallography.

[32]  C. Vieille,et al.  Hyperthermophilic Enzymes: Sources, Uses, and Molecular Mechanisms for Thermostability , 2001, Microbiology and Molecular Biology Reviews.

[33]  M. Adams,et al.  Preparation of genomic DNA from sulfur-dependent hyperthermophilic Archaea , 1995 .

[34]  A. Zehnder,et al.  Characterization of an acetate-decarboxylating, non-hydrogen-oxidizing methane bacterium , 2004, Archives of Microbiology.

[35]  Tohru Kobayashi,et al.  Highly alkaline pectate lyase Pel-4A from alkaliphilic Bacillus sp. strain P-4-N: its catalytic properties and deduced amino acid sequence , 2000, Extremophiles.

[36]  J Schultz,et al.  SMART, a simple modular architecture research tool: identification of signaling domains. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[37]  B Henrissat,et al.  Functional Implications of Structure-Based Sequence Alignment of Proteins in the Extracellular Pectate Lyase Superfamily , 1995, Plant physiology.

[38]  F. Sanger,et al.  DNA sequencing with chain-terminating inhibitors. , 1977, Proceedings of the National Academy of Sciences of the United States of America.

[39]  A. Voragen,et al.  Improvement of the selective depolymerization of pectic substances by chemical β-elimination in aqueous solution , 1992 .