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.
Master
Emma R
ER
Department of Biotechnology, Royal Institute of Technology (KTH), AlbaNova University Center, Stockholm, Sweden.
Rudsander
Ulla J
UJ
Zhou
Weilin
W
Henriksson
Hongbin
H
Divne
Christina
C
Denman
Stuart
S
Wilson
David B
DB
Teeri
Tuula T
TT
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