Overproduction and characterization of seleno-methionine xylanase T-6

Mechaly, Adva; Teplitsky, A.; Belakhov, V. V.; Baasov, Timor; Shoham, G.; Shoham, Yuval

doi:10.1016/s0168-1656(99)00226-6

Cited by 27 publications

(27 citation statements)

References 5 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…However, in contrast to designer cellulosomes, strategies involving multifunctional enzymes are lim- ited to small numbers of enzymes and restricted to suboptimal equimolar ratios of enzymes (7-9, 29, 50). Improvement of xylanase activity has great potential for industrial applications, e.g., prebleaching of kraft pulps and recovering fermentable sugars from natural plant cell wall substrates or directly from hemicellulose (38,47,49,53). Moreover, the worldwide quest to find an alternative to fossil fuels offers major challenges, and studies of designer cellulosomes may eventually provide an approach to meet this challenge (3,4,6).…”

Section: Discussionmentioning

confidence: 99%

Contribution of a Xylan-Binding Module to the Degradation of a Complex Cellulosic Substrate by Designer Cellulosomes

Moraïs

Barak

Caspi

et al. 2010

Appl Environ Microbiol

Self Cite

View full text Add to dashboard Cite

Conversion of components of the Thermobifida fusca free-enzyme system to the cellulosomal mode using the designer cellulosome approach can be employed to discover the properties and inherent advantages of the cellulosome system. In this article, we describe the conversion of the T. fusca xylanases Xyn11A and Xyn10B and their synergistic interaction in the free state or within designer cellulosome complexes in order to enhance specific degradation of hatched wheat straw as a model for a complex cellulosic substrate. Endoglucanase Cel5A from the same bacterium and its recombinant dockerin-containing chimera were also studied for their combined effect, together with the xylanases, on straw degradation. Synergism was demonstrated when Xyn11A was combined with Xyn10B and/or Cel5A, and ϳ1.5-fold activity enhancements were achieved by the designer cellulosome complexes compared to the free wild-type enzymes. These improvements in activity were due to both substrate-targeting and proximity effects among the enzymes contained in the designer cellulosome complexes. The intrinsic cellulose/xylan-binding module (XBM) of Xyn11A appeared to be essential for efficient substrate degradation. Indeed, only designer cellulosomes in which the XBM was maintained as a component of Xyn11A achieved marked enhancement in activity compared to the combination of wild-type enzymes. Moreover, integration of the XBM in designer cellulosomes via a dockerin module (separate from the Xyn11A catalytic module) failed to enhance activity, suggesting a role in orienting the parent xylanase toward its preferred polysaccharide component of the complex wheat straw substrate. The results provide novel mechanistic insight into the synergistic activity of designer cellulosome components on natural plant cell wall substrates.Thermobifida fusca is an aerobic thermophilic soil bacterium with strong cellulolytic activity (52). The T. fusca enzyme system is an extensively studied free cellulase system in which nearly all of the cellulolytic enzymes have been fully characterized, from the individual enzyme sequences to the threedimensional structures, as well as the biochemical activities of the native and recombinant proteins. The genome sequence has been published (36), and the number and types of carbohydrate-active enzymes produced by the organism are known. This actinomycete produces six different cellulases that have been well studied (29,31,32,50,52). T. fusca also has the ability to grow on xylan and produces several enzymes involved in xylan degradation, such as xylanases, ␤-xylosidase, ␣-L-arabinofuranosidase, and acetylesterases (1, 21).Previous research has suggested that the multienzyme cellulosome complex from Clostridium thermocellum is far more efficient than free cellulase systems that were tested in degrading polysaccharides (33). The cellulosome system is characterized by the strong bimodular interaction between the cohesin and dockerin modules that integrates the various enzymes into the complex (5,35,55). Scaffoldin subunits (nonenzymatic ...

show abstract

Section: Discussionmentioning

confidence: 99%

Contribution of a Xylan-Binding Module to the Degradation of a Complex Cellulosic Substrate by Designer Cellulosomes

Moraïs

Barak

Caspi

et al. 2010

Appl Environ Microbiol

Self Cite

View full text Add to dashboard Cite

show abstract

“…For these reasons, we decided to establish a 'fail-safe' model system, which involved the design of a 'universal' carrier protein for the dockerin. For this purpose, we chose xylanase T6 from Geobacillus stearothermophilus, which is known for its exceptionally high propensity towards expression in E. coli host cell systems (Lapidot et al, 1996;Mechaly et al, 2000a). G. stearothermophilus does not produce a cellulosome and the native xylanase-T6 does not bear a dockerin.…”

Section: Preparation and Purification Of Fusion Proteinsmentioning

confidence: 99%

Matching fusion protein systems for affinity analysis of two interacting families of proteins: the cohesin–dockerin interaction

Barak

Handelsman

Nakar

et al. 2005

J of Molecular Recognition

Self Cite

158

View full text Add to dashboard Cite

Cellulosomes are multi-enzyme complexes that orchestrate the efficient degradation of cellulose and related plant cell wall polysaccharides. The complex is maintained by the high-affinity protein-protein interaction between two complementary modules: the cohesin and the dockerin. In order to characterize the interaction between different cohesins and dockerins, we have developed matching fusion-protein systems, which harbor either the cohesin or the dockerin component. For this purpose, corresponding plasmid cassettes were designed, which encoded for the following carrier proteins: (i) a thermostable xylanase with an appended His-tag; and (ii) a highly stable cellulose-binding module (CBM). The resultant xylanase-dockerin and CBM-cohesin fusion products exhibited high expression levels of soluble protein. The expressed, affinity-purified proteins were extremely stable, and the functionality of the cohesin or dockerin component was retained. The fusion protein system was used to establish a sensitive and reliable, semi-quantitative enzyme-linked affinity assay for determining multiple samples of cohesin-dockerin interactions in microtiter plates. A variety of cohesin-dockerin systems, which had been examined previously using other methodologies, were revisited applying the affinity-based enzyme assay, the results of which served to verify the validity of the approach.

show abstract

“…Top, a stereoview of the electron density section around the active site of the E285N mutant in complex with the intact aldotetraouronic acid substrate and the nucleophilic water molecule (contour level of 3.5; green, protein and water; blue, bound substrate). Bottom, a stereoview of the in the methionine auxotrophic Escherichia coli strain B834(DE3), essentially as described previously (17), and protein purification proceeded as for the wild type enzyme. ␣-Glucuronidase activity was determined by measuring the release of MeGlcA from aldotetraouronic acid using the Milner and Avigad assay for uronic acids (18), as described before (16).…”

Section: Figmentioning

confidence: 99%

“…The glycosidic bond is one of the most stable bonds in nature, with a half-life of over 5 million years; glycosidases can accelerate the hydrolysis of these bonds by more than 10 17 -fold (5). The key elements in this catalysis are the position of the catalytic residues and the distortion of the sugar ring so as to allow the stabilization of an oxocarbenium ion-like transition state (6).…”

mentioning

confidence: 99%

Crystal Structures of Geobacillus stearothermophilus α-Glucuronidase Complexed with Its Substrate and Products

Golan

Shallom

Teplitsky

et al. 2004

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

␣-Glucuronidases cleave the ␣-1,2-glycosidic bond between 4-O-methyl-D-glucuronic acid and short xylooligomers as part of the hemicellulose degradation system. To date, all of the ␣-glucuronidases are classified as family 67 glycosidases, which catalyze the hydrolysis via the investing mechanism. Here we describe several high resolution crystal structures of the ␣-glucuronidase (AguA) from Geobacillus stearothermophilus, in complex with its substrate and products. In the complex of AguA with the intact substrate, the 4-O-methyl-D-glucuronic acid sugar ring is distorted into a half-chair conformation, which is closer to the planar conformation required for the oxocarbenium ion-like transition state structure. In the active site, a water molecule is coordinated between two carboxylic acids, in an appropriate position to act as a nucleophile. From the structural data it is likely that two carboxylic acids, Asp 364 and Glu 392 , activate together the nucleophilic water molecule. The loop carrying the catalytic general acid Glu 285 cannot be resolved in some of the structures but could be visualized in its "open" and "closed" (catalytic) conformations in other structures. The protonated state of Glu 285 is presumably stabilized by its proximity to the negative charge of the substrate, representing a new variation of substrate-assisted catalysis mechanism.

show abstract

Overproduction and characterization of seleno-methionine xylanase T-6

Cited by 27 publications

References 5 publications

Contribution of a Xylan-Binding Module to the Degradation of a Complex Cellulosic Substrate by Designer Cellulosomes

Contribution of a Xylan-Binding Module to the Degradation of a Complex Cellulosic Substrate by Designer Cellulosomes

Matching fusion protein systems for affinity analysis of two interacting families of proteins: the cohesin–dockerin interaction

Crystal Structures of Geobacillus stearothermophilus α-Glucuronidase Complexed with Its Substrate and Products

Contact Info

Product

Resources

About