Biochemical characterization of Thermotoga maritima endoglucanase Cel74 with and without a carbohydrate binding module (CBM).
The genome of the hyperthermophilic bacterium Thermotoga maritima (Tm) encodes at least eight glycoside hydrolases with putative signal peptides; the biochemical characteristics of seven of these have been reported previously. The eighth, Tm Cel74, is encoded by an open reading frame of 2124 bp corresponding to a polypeptide of 79 kDa with a signal peptide at the amino-terminus. The gene (lacking the signal peptide) encoding Tm Cel74 was expressed as a 77 kDa monomeric polypeptide in Escherichia coli and found to be optimally active at pH 6, 90 degrees C, with a melting temperature of approximately 105 degrees C. The cel74 gene was previously found to be induced during T. maritima growth on a variety of polysaccharides, including barley glucan, carboxymethyl cellulose (CMC), glucomannan, galactomannan and starch. However, while Tm Cel74 was most active towards barley glucan and to a lesser extent CMC, glucomannan and tamarind (xyloglucan), no activity was detected on other glycans, including galactomannan, laminarin and starch. Also, Tm Cel74 did not contain a carbohydrate binding module (CBM), versions of which have been identified in the amino acid sequences of other family 74 enzymes. As such, a CBM associated with a chitinase in another hyperthermophile, Pyrococcus furiosus, was used to create a fusion protein that was active on crystalline cellulose; Tm Cel74 lacked activity on this substrate. Based on the cleavage pattern determined for Tm Cel74 on glucan-based substrates, this enzyme likely initiates recruitment of carbohydrate carbon and energy sources by creating oligosaccharides that are transported into the cell for further processing.
Chhabra
Swapnil R
SR
Department of Chemical Engineering, North Carolina State University, Stinson Drive, Raleigh, NC 27695-7905, USA.
Kelly
Robert M
RM
D000595:Amino Acid Sequence; D001665:Binding Sites; D050260:Carbohydrate Metabolism; D002480:Cellulase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002482:Cellulose / Q000378:metabolism; D002688:Chitinases / Q000235:genetics; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D020027:Pyrococcus furiosus / Q000201:enzymology; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D016415:Sequence Alignment; D013379:Substrate Specificity; D020124:Thermotoga maritima / Q000201:enzymology*
D011993:Recombinant Fusion Proteins; D002482:Cellulose; D002688:Chitinases; D002480:Cellulase