Recombinant Expression and Enzymatic Characterization of PttCel9A, a KOR Homologue from Populus tremula x tremuloides

• Published in: Biochemistry (Easton) Vol. 43; no. 31; pp. 10080 - 10089
• Main Authors: Master, Emma R, Rudsander, Ulla J, Zhou, Weilin, Henriksson, Hongbin, Divne, Christina, Denman, Stuart, Wilson, David B, Teeri, Tuula T
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 10.08.2004
• Subjects: Amino Acid Sequence; Arabidopsis - enzymology; Bioengineering; Bioteknik; 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; TECHNOLOGY; TEKNIKVETENSKAP; Tetroses - metabolism; Zinc Compounds - metabolism; CULTURED POPLAR CELLS; AGROBACTERIUM-TUMEFACIENS; ENDO-1; CELLULOSE SYNTHESIS; ARABIDOPSIS-THALIANA; IN-GEL DIGESTION; CRYSTAL-STRUCTURE; 4-BETA-GLUCANASE; KORRIGAN; PROTEINS; THERMOMONOSPORA-FUSCA
• ISSN: 0006-2960; 1520-4995; 1520-4995
• DOI: 10.1021/bi049453x

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, Δ1 - 105PttCel9A, 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. Δ1 - 105PttCel9A hydrolyzed a comparatively narrow range of polymeric substrates, and the preferred substrate was (carboxymethyl)cellulose 4M. Moreover, Δ1 - 105PttCel9A 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 °C and pH 6.0, the k cat for cellohexaose of Δ1 - 105PttCel9A, 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 Δ1 - 105PttCel9A 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.