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

ER Master, UJ Rudsander, WL Zhou, H Henriksson, C Divne, S Denman, DB Wilson, TT Teeri*

*Corresponding author for this work

Research output: Contribution to journalArticleScientificpeer-review

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 30degreesC and pH 6.0, the k(cat) 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 (k(cat)/K-m) 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.

Original languageEnglish
Pages (from-to)10080-10089
Number of pages10
JournalBiochemistry
Volume43
Issue number31
DOIs
Publication statusPublished - 10 Aug 2004
MoE publication typeA1 Journal article-refereed

Keywords

  • CULTURED POPLAR CELLS
  • IN-GEL DIGESTION
  • CELLULOSE SYNTHESIS
  • THERMOMONOSPORA-FUSCA
  • ARABIDOPSIS-THALIANA
  • AGROBACTERIUM-TUMEFACIENS
  • CRYSTAL-STRUCTURE
  • ENDO-1,4-BETA-GLUCANASE
  • PROTEINS
  • KORRIGAN

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