Cloning and characterization of the glucooligosaccharide catabolic pathway beta-glucan glucohydrolase and cellobiose phosphorylase in the marine hyperthermophile Thermotoga neapolitana.
Characterization in Thermotoga neapolitana of a catabolic gene cluster encoding two glycosyl hydrolases, 1,4-beta-D-glucan glucohydrolase (GghA) and cellobiose phosphorylase (CbpA), and the apparent absence of a cellobiohydrolase (Cbh) suggest a nonconventional pathway for glucan utilization in Thermotogales. GghA purified from T. neapolitana is a 52.5-kDa family 1 glycosyl hydrolase with optimal activity at pH 6.5 and 95 degrees C. GghA releases glucose from soluble glucooligomers, with a preference for longer oligomers: k(cat)/K(m) values are 155.2, 76.0, and 9.9 mM(-1) s(-1) for cellotetraose, cellotriose, and cellobiose, respectively. GghA has broad substrate specificity, with specific activities of 236 U/mg towards cellobiose and 251 U/mg towards lactose. With p-nitrophenyl-beta-glucoside as the substrate, GghA exhibits biphasic kinetic behavior, involving both substrate- and end product-directed activation. Its capacity for transglycosylation is a factor in this activation. Cloning of gghA revealed a contiguous upstream gene (cbpA) encoding a 93.5-kDa cellobiose phosphorylase. Recombinant CbpA has optimal activity at pH 5.0 and 85 degrees C. It has specific activity of 11.8 U/mg and a K(m) of 1.42 mM for cellobiose, but shows no activity towards other disaccharides or cellotriose. With its single substrate specificity and low K(m) for cellobiose (compared to GghA's K(m) of 28.6 mM), CbpA may be the primary enzyme for attacking cellobiose in Thermotoga spp. By phosphorolysis of cellobiose, CbpA releases one activated glucosyl molecule while conserving one ATP molecule per disaccharide. CbpA is the first hyperthermophilic cellobiose phosphorylase to be characterized.
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Department of Biochemistry and Microbiology, Cook College, Rutgers University, New Brunswick, New Jersey 08901, USA.
McCarthy
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Eveleigh
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Bok
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D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D044643:Glucan 1,4-beta-Glucosidase; D005964:Glucosyltransferases / Q000235:genetics* / Q000378:metabolism*; D016965:Gram-Negative Anaerobic Straight, Curved, and Helical Rods / Q000201:enzymology* / Q000235:genetics; D007700:Kinetics; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D001617:beta-Glucosidase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*
D009844:Oligosaccharides; D011994:Recombinant Proteins; D005964:Glucosyltransferases; C034714:cellobiose phosphorylase; D001617:beta-Glucosidase; D044643:Glucan 1,4-beta-Glucosidase