Cloning and Characterization of the Glucooligosaccharide Catabolic Pathway β-Glucan Glucohydrolase and Cellobiose Phosphorylase in the Marine HyperthermophileThermotoga neapolitana

Abstract

Characterization in Thermotoga neapolitana of a catabolic gene cluster encoding two glycosyl hydrolases, 1,4-␤-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°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-␤-glucoside as the substrate, GghA exhibits biphasic kinetic behavior, involving both substrateand 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°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.To utilize polysaccharides such as glucans to meet carbon and energy requirements, heterotrophic organisms depend on a catabolic pathway involving the interaction of multiple hydrolytic enzymes, transporter complexes, and regulatory systems coordinating gene expression of pathway-specific proteins (41). During hydrolysis of the homopolymer cellulose, for example, the consortium of catalytic enzymes consists of endoglucanases (1,4-␤-D-glucan 4-glucohydrolase [EC 3.2.1.4]), which randomly hydrolyze internal ␤-1,4 glycosidic bonds; cellobiohydrolases (␤-D-glucan cellobiohydrolase [EC 3.2.1.91]), also referred to as exoglucanases, which remove cellobiose from either the nonreducing or reducing ends of cellooligomers; and ␤-glucosidases (␤-D-glucoside glucohydrolase [EC 3.2.1.21]), which convert cellobiose to glucose. These classes of enzymes have been documented in a number of fungal and bacterial systems (4, 5, 39), while other glucan-catabolyzing enzymes are less well understood.Thermotoga neapolitana, a marine hyperthermophile isolated from geothermally heated biotopes, belongs to the order Thermotogales. T. neapolitana shares with other Thermotogales, specifically Thermotoga maritima, both the capacity to catabolize a wide variety of ␣-and ␤-linked glucans and a fermentative metabolism. While Thermotogales elaborate hydrolases such as amylases, cellulases, glucosidases, galac...

Gene Encoding a Trehalose Phosphorylase fromThermoanaerobacter brockiiATCC 35047

“…Although TPs from G. frondosa and T. brockii catalyze the phosphorolysis of trehalose, their amino acid sequences show only 14z identity. The value is nearly identical to the identity between Thermoanaerobacter TP and other a-G1P-dependent disaccharide phosphorylases; 16z and 17z with cellobiose phosphorylases from Thermotoga neapolitana 14) and Cellvibrio gilvus, 15) respectively; 15z and 14z with sucrose phosphorylases from Leuconostoc mesenteroides 16) and Agrobacterium vitis, 17) respectively. This also supports a relationship between the structure of b-G1P-producing phosphorylases and the anomeric conˆguration through the catalysis.…”

Microbial Cellulose Utilization: Fundamentals and Biotechnology

“…CbP is widely distributed in cellobiose-utilizing bacteria, both anaerobic (9,10,23,249,667,738,740) and aerobic (600), and the enzyme has even been found in the hyperthermophile Thermotoga neopolitana (769). The CbP from Cellvibrio gilvus is particularly interesting in that its wide glucosyl acceptor specificity has permitted the in vitro synthesis of numerous unusual di-and trisaccharides (670).…”

Carbohydrate-induced Differential Gene Expression Patterns in the Hyperthermophilic Bacterium Thermotoga maritima

“…Based on specific gene expression patterns observed during growth on CMC, the biochemical characteristics of TM1524 (Cel12A) (38), TM1525 (38), TM1848 (39), and TM0305 (40), the cellular localization of these proteins (5), and the motifs described, a mechanism for the uptake and utilization of CMC by T. maritima can be proposed (see Fig. 6A).…”