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

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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, 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 kcat 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 (kcat/Km) 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.