Cel9D, an atypical 1,4-beta-D-glucan glucohydrolase from Fibrobacter succinogenes: characteristics, catalytic residues, and synergistic interactions with other cellulases.
The increasing demands of renewable energy have led to the critical emphasis on novel enzymes to enhance cellulose biodegradation for biomass conversion. To identify new cellulases in the ruminal bacterium Fibrobacter succinogenes, a cell extract of cellulose-grown cells was separated by ion-exchange chromatography and cellulases were located by zymogram analysis and identified by peptide mass fingerprinting. An atypical family 9 glycoside hydrolase (GH9), Cel9D, with less than 20% identity to typical GH9 cellulases, was identified. Purified recombinant Cel9D enhanced the production of reducing sugar from acid swollen cellulose (ASC) and Avicel by 1.5- to 4-fold when mixed separately with each of four other glucanases, although it had low activity on these substrates. Cel9D degraded ASC and cellodextrins with a degree of polymerization higher than 2 to glucose with no apparent endoglucanase activity, and its activity was restricted to beta-1-->4-linked glucose residues. It catalyzed the hydrolysis of cellulose by an inverting mode of reaction, releasing glucose from the nonreducing end. Unlike many GH9 cellulases, calcium ions were not required for its function. Cel9D had increased kcat/Km values for cello-oligosaccharides with higher degrees of polymerization. The kcat/Km value for cellohexaose was 2,300 times higher than that on cellobiose. This result indicates that Cel9D is a 1,4-beta-D-glucan glucohydrolase (EC 3.2.1.74) in the GH9 family. Site-directed mutagenesis of Cel9D identified Asp166 and Glu612 as the candidate catalytic residues, while Ser168, which is not present in typical GH9 cellulases, has a crucial structural role. This enzyme has an important role in crystalline cellulose digestion by releasing glucose from accessible cello-oligosaccharides.
Qi
Meng
M
Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario N1G 2W1, Canada.
Jun
Hyun-Sik
HS
Forsberg
Cecil W
CW
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D020134:Catalytic Domain / Q000235:genetics; D044602:Cellulases / Q000235:genetics / Q000378:metabolism*; D002482:Cellulose / Q000031:analogs & derivatives / Q000378:metabolism; D002852:Chromatography, Ion Exchange; D003912:Dextrins / Q000378:metabolism; D045848:Fibrobacter / Q000201:enzymology* / Q000235:genetics; D016680:Genome, Bacterial / Q000235:genetics; D005959:Glucosidases / Q000235:genetics / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D001426:Bacterial Proteins; D003912:Dextrins; D002482:Cellulose; C068994:cellodextrin; D044602:Cellulases; D005959:Glucosidases; C446280:beta-D-glucan glucohydrolase