EndB, a multidomain family 44 cellulase from Ruminococcus flavefaciens 17, binds to cellulose via a novel cellulose-binding module and to another R. flavefaciens protein via a dockerin domain.
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The mechanisms by which cellulolytic enzymes and enzyme complexes in Ruminococcus spp. bind to cellulose are not fully understood. The product of the newly isolated cellulase gene endB from Ruminococcus flavefaciens 17 was purified as a His-tagged product after expression in Escherichia coli and found to be able to bind directly to crystalline cellulose. The ability to bind cellulose is shown to be associated with a novel cellulose-binding module (CBM) located within a region of 200 amino acids that is unrelated to known protein sequences. EndB (808 amino acids) also contains a catalytic domain belonging to glycoside hydrolase family 44 and a C-terminal dockerin-like domain. Purified EndB is also shown to bind specifically via its dockerin domain to a polypeptide of ca. 130 kDa present among supernatant proteins from Avicel-grown R. flavefaciens that attach to cellulose. The protein to which EndB attaches is a strong candidate for the scaffolding component of a cellulosome-like multienzyme complex recently identified in this species (S.-Y. Ding et al., J. Bacteriol. 183:1945-1953, 2001). It is concluded that binding of EndB to cellulose may occur both through its own CBM and potentially also through its involvement in a cellulosome complex. (+info)
Cloning and overexpression of the avi2 gene encoding a major cellulase produced by Humicola insolens FERM BP-5977.
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The avi2 gene encoding Avi2, which is a major cellulase produced by Humicola insolens FERM BP-5977, was cloned and sequenced. Avi2 showed high homology with other family 6 cellulases. The expression vector pNCE4 containing the avi2 gene was constructed, and this strain was transformed using a protoplast method. As a result, the pNCE4 transformant secreted 8-fold more Avi2 than the recipient strain. (+info)
Termite gut symbiotic archaezoa are becoming living metabolic fossils.
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Over the course of several million years, the eukaryotic gut symbionts of lower termites have become adapted to a cellulolytic environment. Up to now it has been believed that they produce nutriments using their own cellulolytic enzymes for the benefit of their termite host. However, we have now isolated two endoglucanases with similar apparent molecular masses of approximately 36 kDa from the not yet culturable symbiotic Archaezoa living in the hindgut of the most primitive Australian termite, Mastotermes darwiniensis. The N-terminal sequences of these cellulases exhibited significant homology to cellulases of termite origin, which belong to glycosyl hydrolase family 9. The corresponding genes were detected not in the mRNA pool of the flagellates but in the salivary glands of M. darwiniensis. This showed that cellulases isolated from the flagellate cells originated from the termite host. By use of a PCR-based approach, DNAs encoding cellulases belonging to glycosyl hydrolase family 45 were obtained from micromanipulated nuclei of the flagellates Koruga bonita and Deltotrichonympha nana. These results indicated that the intestinal flagellates of M. darwiniensis take up the termite's cellulases from gut contents. K. bonita and D. nana possess at least their own endoglucanase genes, which are still expressed, but without significant enzyme activity in the nutritive vacuole. These findings give the impression that the gut Archaezoa are heading toward a secondary loss of their own endoglucanases and that they use exclusively termite cellulases. (+info)
Ruminococcus albus 8 mutants defective in cellulose degradation are deficient in two processive endocellulases, Cel48A and Cel9B, both of which possess a novel modular architecture.
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The cellulolytic bacterium Ruminococcus albus 8 adheres tightly to cellulose, but the molecular biology underpinning this process is not well characterized. Subtractive enrichment procedures were used to isolate mutants of R. albus 8 that are defective in adhesion to cellulose. Adhesion of the mutant strains was reduced 50% compared to that observed with the wild-type strain, and cellulose solubilization was also shown to be slower in these mutant strains, suggesting that bacterial adhesion and cellulose solubilization are inextricably linked. Two-dimensional polyacrylamide gel electrophoresis showed that all three mutants studied were impaired in the production of two high-molecular-mass, cell-bound polypeptides when they were cultured with either cellobiose or cellulose. The identities of these proteins were determined by a combination of mass spectrometry methods and genome sequence data for R. albus 8. One of the polypeptides is a family 9 glycoside hydrolase (Cel9B), and the other is a family 48 glycoside hydrolase (Cel48A). Both Cel9B and Cel48A possess a modular architecture, Cel9B possesses features characteristic of the B(2) (or theme D) group of family 9 glycoside hydrolases, and Cel48A is structurally similar to the processive endocellulases CelF and CelS from Clostridium cellulolyticum and Clostridium thermocellum, respectively. Both Cel9B and Cel48A could be recovered by cellulose affinity procedures, but neither Cel9B nor Cel48A contains a dockerin, suggesting that these polypeptides are retained on the bacterial cell surface, and recovery by cellulose affinity procedures did not involve a clostridium-like cellulosome complex. Instead, both proteins possess a single copy of a novel X module with an unknown function at the C terminus. Such X modules are also present in several other R. albus glycoside hydrolases and are phylogentically distinct from the fibronectin III-like and X modules identified so far in other cellulolytic bacteria. (+info)
Dietary lignins are precursors of mammalian lignans in rats.
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The mammalian lignans enterolactone (ENL) and enterodiol, commonly found in human plasma and urine, are phytoestrogens that may contribute to the prevention of breast cancer and coronary heart disease. They are formed by the conversion of dietary precursors such as secoisolariciresinol and matairesinol lignans by the colonic microflora. The identification of lignins, cell-wall polymers structurally related to lignans, as precursors of mammalian lignans is reported here for the first time. In study 1, rats were fed rye or wheat bran (15% diet) for 5 d. Untreated brans and brans extracted with solvents to remove lignans were compared. ENL was estimated in urine samples collected for 24 h by time-resolved fluoroimmunoassay. ENL urinary excretion was reduced from 18.6 to 5.3 nmol/d (n=8; P<0.001) when lignans were removed from rye bran and from 30.5 to 6.2 nmol/d (P<0.001) when they were removed from wheat bran. These results suggest that lignins, embedded in the cell wall and retained in the bran during solvent extraction, account for 26-32% of the ENL formed from cereal brans. In study 2, rats were fed a deuterated synthetic lignin (0.2% diet) together with wheat bran (15%) for 3 d. The detection of deuterated ENL by LC-tandem MS in urine (20 nmol/d) clearly confirms the conversion of lignin into mammalian lignans. More research is warranted to determine the bioavailability of lignins in the human diet. (+info)
The catalytic module of Hypocrea jecorina (previously Trichoderma reesei) Cel7B was homologously expressed by transformation of strain QM9414. Post-translational modifications in purified Cel7B preparations were analysed by enzymatic digestions, high performance chromatography, mass spectrometry and site-directed mutagenesis. Of the five potential sites found in the wild-type enzyme, only Asn56 and Asn182 were found to be N-glycosylated. GlcNAc(2)Man(5) was identified as the predominant N-glycan, although lesser amounts of GlcNAc(2)Man(7) and glycans carrying a mannophosphodiester bond were also detected. Repartition of neutral and charged glycan structures over the two glycosylation sites mainly accounts for the observed microheterogeneity of the protein. However, partial deamidation of Asn259 and a partially occupied O-glycosylation site give rise to further complexity in enzyme preparations. (+info)
Fungal cell wall chitinases and glucanases.
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The fungal cell wall is a complex structure composed of chitin, glucans and other polymers, and there is evidence of extensive cross-linking between these components. The wall structure is highly dynamic, changing constantly during cell division, growth and morphogenesis. Hydrolytic enzymes, closely associated with the cell wall, have been implicated in the maintenance of wall plasticity and may have roles during branching and cross-linking of polymers. Most fungal cell wall hydrolases identified to date have chitinase or glucanase activity and this short article reviews the apparent functions of these enzymes in unicellular and filamentous fungi, and the mechanisms that regulate enzyme activity in yeasts. (+info)
Structural, thermodynamic, and kinetic analyses of tetrahydrooxazine-derived inhibitors bound to beta-glucosidases.
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The understanding of transition state mimicry in glycoside hydrolysis is increasingly important both in the quest for novel specific therapeutic agents and for the deduction of enzyme function and mechanism. To aid comprehension, inhibitors can be characterized through kinetic, thermodynamic, and structural dissection to build an "inhibition profile." Here we dissect the binding of a tetrahydrooxazine inhibitor and its derivatives, which display Ki values around 500 nm. X-ray structures with both a beta-glucosidase, at 2 A resolution, and an endoglucanase at atomic (approximately 1 A) resolution reveal similar interactions between the tetrahydrooxazine inhibitor and both enzymes. Kinetic analyses reveal the pH dependence of kcat/Km and 1/Ki with both enzyme systems, and isothermal titration calorimetry unveils the enthalpic and entropic contributions to beta-glucosidase inhibition. The pH dependence of enzyme activity mirrored that of 1/Ki in both enzymes, unlike the cases of isofagomine and 1-deoxynojirimycin that have been characterized previously. Calorimetric dissection reveals a large favorable enthalpy that is partially offset by an unfavorable entropy upon binding. In terms of the similar profile for the pH dependence of 1/Ki and the pH dependence of kcat/Km, the significant enthalpy of binding when compared with other glycosidase inhibitors, and the tight binding at the optimal pH of the enzymes tested, tetrahydrooxazine and its derivatives are a significantly better class of glycosidase inhibitor than previously assumed. (+info)