Thermotoga neapolitana bgIB gene, upstream of lamA, encodes a highly thermostable β-glucosidase that is a laminaribiase

Zverlov, Vladimir V.; Volkov, Ilia Y.; Velikodvorskaya, Tatjana V.; Schwarz, Wolfgang H.

doi:10.1099/00221287-143-11-3537

Cited by 49 publications

(36 citation statements)

References 15 publications

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“…A schematic conversion of cellooligomers to activated glucosyl molecules. Enzymes indicated are CelA and CelB, endoglucanases (7) active in or on the toga; GghA, glucan glucohydrolase, and CbpA, cellobiose phosphorylase, intracellular enzymes (this work); and LamA and BglB, laminarinase and ␤-glucosidase, respectively (45,46). of the process of acquisition of carbon and energy by Thermotogales.…”

Section: Discussionmentioning

confidence: 99%

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

et al. 2000

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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...

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Section: Discussionmentioning

confidence: 99%

“…6). Other potential components of this catabolic pathway might include a laminarinase (LamA) and a laminaribiase/␤-glucosidase (BglB), which act on both ␤-1,3 and ␤-1,4 linkages (45,46).…”

Section: Discussionmentioning

confidence: 99%

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

et al. 2000

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“…The b-1,3-glucanases produce a characteristic pattern of b-1,3-oligosaccharides, which are further degraded by b-glucosidases. In contrast to the highly specific b-glucanases, b-glucosidases degrade laminarioligosaccharides (b-1,3) as well as cello-oligosaccharides (b-1,4) (Gräbnitz, et al 1989;Zverlov et al, 1997b).…”

Section: Introductionmentioning

confidence: 99%

Lic16A of Clostridium thermocellum, a non-cellulosomal, highly complex endo-β-1,3-glucanase bound to the outer cell surface

Fuchs¹,

Zverlov

Velikodvorskaya

et al. 2003

Microbiology

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Clostridium thermocellum produces one major b-1,3-glucanase. Genomic DNA fragments containing the gene were cloned from two strains, DSM1237T (6848 bp) and F7 (9766 bp).Overlapping sequences were 99?9 % identical. The nucleotide sequences contained reading frames for a putative transposase, endo-b-1,3-1,4-glucanase CelC, a putative transcription regulator of the LacI type, b-1,3-glucanase Lic16A and a putative membrane protein. The licA genes of both strains encoded an identical protein of 1324 aa with a calculated molecular mass of 148 kDa. Lic16A is an unusually complex protein consisting of a leader peptide, a threefold repeat of an S-layer homologous module (SLH), an unknown module, a catalytic module of glycosyl hydrolase family 16 and a fourfold repeat of a carbohydrate-binding module of family CBM4a. The recombinant Lic16A protein was characterized as an endo-1,3(4)-b-glucanase with a specific activity of 2680 and 340 U mg 21 and a K m of 0?94 and 2?1 mg ml 21 towards barley b-glucan and laminarin, respectively. It was specific for b-glucans containing b-1,3-linkages with an optimum temperature of 70˚C at pH 6?0. The N-terminal SLH modules were cleaved from the protein as well in Escherichia coli as in C. thermocellum, but nevertheless bound tightly to the rest of the protein. Lic16A was located on the cell surface from which it could be purified after fractionated solubilization. Its inducible production allowed C. thermocellum to grow on b-1,3-or b-1,3-1,4-glucan.

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“…LamA (endo-␤-(1,3)-glucanase) was a recombinant protein from Thermotoga neapolitana expressed in Escherichia coli (a kind gift from Dr. Vladimir V. Zverlov, Institute of Molecular Genetics, Russian Academy of Science, Moscow, Russia) (27). Endo-␤-(1,6)-glucanase from Trichoderma harzianum was expressed in Pichia pastoris (a kind gift from Prof. Stanley Brul, Swammerdam Institute for Life Sciences, Amsterdam, The Netherlands) (28).…”

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confidence: 99%

Cell Wall β-(1,6)-Glucan of Saccharomyces cerevisiae

Aimanianda

Clavaud

Simenel

et al. 2009

Journal of Biological Chemistry

120

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Despite its essential role in the yeast cell wall, the exact composition of the ␤-(1,6)-glucan component is not well characterized. While solubilizing the cell wall alkali-insoluble fraction from a wild type strain of Saccharomyces cerevisiae using a recombinant ␤-(1,3)-glucanase followed by chromatographic characterization of the digest on an anion exchange column, we observed a soluble polymer that eluted at the end of the solvent gradient run. Further characterization indicated this soluble polymer to have a molecular mass of ϳ38 kDa and could be hydrolyzed only by ␤-(1,6)-glucanase. Gas chromatographymass spectrometry and NMR ( 1 H and 13 C) analyses confirmed it to be a ␤-(1,6)-glucan polymer with, on average, branching at every fifth residue with one or two ␤-(1,3)-linked glucose units in the side chain. This polymer peak was significantly reduced in the corresponding digests from mutants of the kre genes (kre9 and kre5) that are known to play a crucial role in the ␤-(1,6)-glucan biosynthesis. In the current study, we have developed a biochemical assay wherein incubation of UDP-[ 14 C]glucose with permeabilized S. cerevisiae yeasts resulted in the synthesis of a polymer chemically identical to the branched ␤-(1,6)-glucan isolated from the cell wall. Using this assay, parameters essential for ␤-(1,6)-glucan synthetic activity were defined.

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Thermotoga neapolitana bgIB gene, upstream of lamA, encodes a highly thermostable β-glucosidase that is a laminaribiase

Cited by 49 publications

References 15 publications

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

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

Lic16A of Clostridium thermocellum, a non-cellulosomal, highly complex endo-β-1,3-glucanase bound to the outer cell surface

Cell Wall β-(1,6)-Glucan of Saccharomyces cerevisiae

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