| Recombinant expression and enzymatic characterization of PttCel9A, a KOR homologue from Populus tremula x tremuloides. | |
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MedLine Citation:
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PMID: 15287736 Owner: NLM Status: MEDLINE |
Abstract/OtherAbstract:
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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. |
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Authors:
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Emma R Master; Ulla J Rudsander; Weilin Zhou; Hongbin Henriksson; Christina Divne; Stuart Denman; David B Wilson; Tuula T Teeri |
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Publication Detail:
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Type: Comparative Study; Journal Article; Research Support, Non-U.S. Gov't |
Journal Detail:
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Title: Biochemistry Volume: 43 ISSN: 0006-2960 ISO Abbreviation: Biochemistry Publication Date: 2004 Aug |
Date Detail:
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Created Date: 2004-08-03 Completed Date: 2004-09-30 Revised Date: 2006-11-15 |
Medline Journal Info:
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Nlm Unique ID: 0370623 Medline TA: Biochemistry Country: United States |
Other Details:
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Languages: eng Pagination: 10080-9 Citation Subset: IM |
Affiliation:
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Department of Biotechnology, Royal Institute of Technology (KTH), AlbaNova University Center, Stockholm, Sweden. |
Export Citation:
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| MeSH Terms | |
Descriptor/Qualifier:
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Amino Acid Sequence Arabidopsis / enzymology Calcium Chloride / metabolism Cations, Divalent / metabolism Cellulose / analogs & derivatives*, metabolism Chlorides / metabolism Hydrogen-Ion Concentration Hydrolysis Kinetics Molecular Sequence Data Mutagenesis, Site-Directed N-Glycosyl Hydrolases / biosynthesis, genetics*, isolation & purification, metabolism* Oligosaccharides / metabolism Pichia / enzymology, genetics Plant Proteins / biosynthesis, genetics*, isolation & purification, metabolism* Polymers / metabolism Populus / enzymology*, genetics* Recombinant Proteins / biosynthesis, chemistry* Sequence Homology, Amino Acid* Substrate Specificity Tetroses / metabolism Zinc Compounds / metabolism |
| Chemical | |
Reg. No./Substance:
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0/Cations, Divalent; 0/Chlorides; 0/Oligosaccharides; 0/Plant Proteins; 0/Polymers; 0/Recombinant Proteins; 0/Tetroses; 0/Zinc Compounds; 10043-52-4/Calcium Chloride; 2478-35-5/cellohexaose; 38819-01-1/cellotetraose; 7646-85-7/zinc chloride; 9004-34-6/Cellulose; EC 3.2.2.-/N-Glycosyl Hydrolases |
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine
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