Recombinant expression and enzymatic characterization of PttCel9A, a KOR homologue from Populus tremula x tremuloides.
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.
Department of Biotechnology, Royal Institute of Technology (KTH), AlbaNova University Center, Stockholm, Sweden.
Emma R
ER
Master
Ulla J
UJ
Rudsander
Weilin
W
Zhou
Hongbin
H
Henriksson
Christina
C
Divne
Stuart
S
Denman
David B
DB
Wilson
Tuula T
TT
Teeri
D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000201:enzymology; D002122:Calcium Chloride / Q000378:metabolism; D002413:Cations, Divalent / Q000378:metabolism; D002482:Cellulose / Q000031:analogs & derivatives* / Q000378:metabolism; D002712:Chlorides / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009699:N-Glycosyl Hydrolases / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D009844:Oligosaccharides / Q000378:metabolism; D010843:Pichia / Q000201:enzymology / Q000235:genetics; D010940:Plant Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D011108:Polymers / Q000378:metabolism; D032107:Populus / Q000201:enzymology* / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry*; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013780:Tetroses / Q000378:metabolism; D017967:Zinc Compounds / Q000378:metabolism
D002413:Cations, Divalent; D002712:Chlorides; D009844:Oligosaccharides; D010940:Plant Proteins; D011108:Polymers; D011994:Recombinant Proteins; D013780:Tetroses; D017967:Zinc Compounds; C099423:cellohexaose; C048468:cellotetraose; C016837:zinc chloride; D002482:Cellulose; D009699:N-Glycosyl Hydrolases; D002122:Calcium Chloride