Recombinant expression and enzymatic characterization of PttCel9A, a KOR homologue from Populus tremula x tremuloides

Biochemistry. 2004 Aug 10;43(31):10080-9. doi: 10.1021/bi049453x.

Abstract

PttCel9A is a membrane-bound, family 9 glycosyl hydrolase from Populus tremula x tremuloides that is upregulated during secondary cell wall synthesis. The catalytic domain of PttCel9A, Delta(1-105)PttCel9A, was purified, and its activity was compared to TfCel9A and TfCel9B from Thermobifida fusca. Since aromatic amino acids involved in substrate binding at subsites -4, -3, and -2 are missing in PttCel9A, the activity of TfCel9A mutant enzymes W256S, W209A, and W313G was also investigated. Delta(1-105)PttCel9A hydrolyzed a comparatively narrow range of polymeric substrates, and the preferred substrate was (carboxymethyl)cellulose 4M. Moreover, Delta(1-105)PttCel9A did not hydrolyze oligosaccharides shorter than cellopentaose, whereas TfCel9A and TfCel9B hydrolyzed cellotetraose and cellotriose, respectively. These data suggest that the preferred substrates of PttCel9A are long, low-substituted, soluble cellulosic polymers. At 30 degrees C and pH 6.0, the kcat for cellohexaose of Delta(1-105)PttCel9A, TfCel9A, and TfCel9B were 0.023 +/- 0.001, 16.9 +/- 2.0, and 1.3 +/- 0.2, respectively. The catalytic efficiency (kcat/Km) of TfCel9B was 39% of that of TfCel9A, whereas the catalytic efficiency of Delta(1-105)PttCel9A was 0.04% of that of TfCel9A. Removing tryptophan residues at subsites -4, -3, and -2 decreased the efficiency of cellohexaose hydrolysis by TfCel9A. Mutation of W313 to G had the most drastic effect, producing a mutant enzyme with 1% of the catalytic efficiency of TfCel9A. The apparent narrow substrate range and catalytic efficiency of PttCel9A are correlated with a lack of aromatic amino acids in the substrate binding cleft and may be necessary to prevent excessive hydrolysis of cell wall polysaccharides during cell wall formation.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Arabidopsis / enzymology
  • Calcium Chloride / metabolism
  • Cations, Divalent / metabolism
  • Cellulose / analogs & derivatives*
  • Cellulose / metabolism
  • Chlorides / metabolism
  • Hydrogen-Ion Concentration
  • Hydrolysis
  • Kinetics
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • N-Glycosyl Hydrolases / biosynthesis
  • N-Glycosyl Hydrolases / genetics*
  • N-Glycosyl Hydrolases / isolation & purification
  • N-Glycosyl Hydrolases / metabolism*
  • Oligosaccharides / metabolism
  • Pichia / enzymology
  • Pichia / genetics
  • Plant Proteins / biosynthesis
  • Plant Proteins / genetics*
  • Plant Proteins / isolation & purification
  • Plant Proteins / metabolism*
  • Polymers / metabolism
  • Populus / enzymology*
  • Populus / genetics*
  • Recombinant Proteins / biosynthesis
  • Recombinant Proteins / chemistry*
  • Sequence Homology, Amino Acid*
  • Substrate Specificity
  • Tetroses / metabolism
  • Zinc Compounds / metabolism

Substances

  • Cations, Divalent
  • Chlorides
  • Oligosaccharides
  • Plant Proteins
  • Polymers
  • Recombinant Proteins
  • Tetroses
  • Zinc Compounds
  • cellohexaose
  • cellotetraose
  • zinc chloride
  • Cellulose
  • N-Glycosyl Hydrolases
  • Calcium Chloride