Dataset Information

Structural basis of product inhibition by arabinose and xylose of the thermostable GH43 ?-1,4-xylosidase from Geobacillus thermoleovorans IT-08.

ABSTRACT: Complete degradation of the xylan backbone of hemicellulosic plant cell walls requires the synergistic action of endo-xylanases and ?-1,4-xylosidases. While endo-xylanases produce xylooligosaccharides from xylan, ?-1,4-xylosidases degrade the xylooligosaccharides into xylose monomers. The glycoside hydrolase family 43 ?-1,4-xylosidase from Geobacillus thermoleovorans IT-08 is a promising, heat stable catalyst for the saccharification of hemicellulosic material into simple fermentable sugars, but it is competitively inhibited by its products arabinose and xylose. As a first step to help overcome this problem, we elucidated crystal structures of the enzyme in the unliganded form and with bound products, at 1.7-2.0 Å resolution. The structures are very similar to those of other enzymes belonging to glycoside hydrolase family 43. Unexpectedly, the monosaccharides are bound in very different ways. Arabinose preferentially binds in subsite -1, while xylose exclusively interacts with subsite +1. These structures and sugar binding preferences suggest ways for improving the catalytic performance of the enzyme by rational mutational design.

SUBMITTER: Rohman A

PROVIDER: S-EPMC5919610 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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Publications

Structural basis of product inhibition by arabinose and xylose of the thermostable GH43 β-1,4-xylosidase from Geobacillus thermoleovorans IT-08.

Rohman Ali A van Oosterwijk Niels N Puspaningsih Ni Nyoman Tri NNT Dijkstra Bauke W BW

PloS one 20180426 4

Complete degradation of the xylan backbone of hemicellulosic plant cell walls requires the synergistic action of endo-xylanases and β-1,4-xylosidases. While endo-xylanases produce xylooligosaccharides from xylan, β-1,4-xylosidases degrade the xylooligosaccharides into xylose monomers. The glycoside hydrolase family 43 β-1,4-xylosidase from Geobacillus thermoleovorans IT-08 is a promising, heat stable catalyst for the saccharification of hemicellulosic material into simple fermentable sugars, but ...[more]

PMID: 29698436

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Project description:BACKGROUND: Complete enzymatic hydrolysis of xylan to xylose requires the action of endoxylanase and ?-xylosidase. ?-xylosidases play an important part in hydrolyzing xylo-oligosaccharides to xylose. Thermostable ?-xylosidases have been a focus of attention as industrially important enzymes due to their long shelf life and role in the relief of end-product inhibition of xylanases caused by xylo-oligosaccharides. Therefore, a highly thermostable ?-xylosidase with high specific activity has significant potential in lignocellulose bioconversion. RESULTS: A gene encoding a highly thermostable GH39 ?-xylosidase was cloned from Geobacillus sp. strain WSUCF1 and expressed in Escherichia coli. Recombinant ?-xylosidase was active over a wide range of temperatures and pH with optimum temperature of 70 °C and pH 6.5. It exhibited very high thermostability, retaining 50% activity at 70 °C after 9 days. WSUCF1 ?-xylosidase is more thermostable than ?-xylosidases reported from other thermophiles (growth temperature ? 70 °C). Specific activity was 133 U/mg when incubated with p-nitrophenyl xylopyranoside, with Km and Vmax values of 2.38 mM and 147 U/mg, respectively. SDS-PAGE analysis indicated that the recombinant enzyme had a mass of 58 kDa, but omitting heating prior to electrophoresis increased the apparent mass to 230 kDa, suggesting the enzyme exists as a tetramer. Enzyme exhibited high tolerance to xylose, retained approximately 70% of relative activity at 210 mM xylose concentration. Thin layer chromatography showed that the enzyme had potential to convert xylo-oligomers (xylobiose, triose, tetraose, and pentaose) into fermentable xylose. WSUCF1 ?-xylosidase along with WSUCF1 endo-xylanase synergistically converted the xylan into fermentable xylose with more than 90% conversion. CONCLUSIONS: Properties of the WSUCF1 ?-xylosidase i.e. high tolerance to elevated temperatures, high specific activity, conversion of xylo-oligomers to xylose, and resistance to inhibition from xylose, make this enzyme potentially suitable for various biotechnological applications.

Project description:BackgroundMaltogenic amylases belong to a subclass of cyclodextrin-hydrolyzing enzymes and hydrolyze cyclodextrins more efficiently than starch unlike typical α-amylases. Several bacterial malto-genic amylases with temperature optima of 40-60°C have been previously characterized. The thermo-adaption, substrate preferences and transglycosylation aspects of extremely thermostable bacterial maltogenic amylases have not yet been reported.Methodology/principal findingsThe recombinant monomeric and dimeric forms of maltogenic α-amylase (Gt-Mamy) of the extremely thermophilic bacterium Geobacillus thermoleovorans are of 72.5 and 145 kDa, which are active optimally at 80°C. Extreme thermostability of this enzyme has been explained by analyzing far-UV CD spectra. Dimerization increases T1/2 of Gt-Mamy from 8.2 h to 12.63 h at 90°C and mediates its enthalpy-driven conformational thermostabilization. Furthermore, dime-rization regulates preferential substrate binding of the enzyme. The substrate preference switching of Gt-Mamy upon dimerization has been confirmed from the substrate-binding affinities of the enzyme for various high and low molecular weight substrates. There is an alteration in Km and substrate hydrolysis efficiency (Vmax/Km) of the enzyme (for cyclodex-trins/starch) upon dimerization. N-terminal truncation indicated the role of N-terminal 128 amino acids in the thermostabilization and modulation of substrate-binding affinity. This has been confirmed by molecular docking of β-cyclodextrin to Gt-Mamy that indicated the requirement of homodimer formation by the interaction of a few N-terminal residues of chain A with the catalytic residues of (α/β)8 barrel of chain B and vice-versa for stable cyclodextrin binding. Site directed mutagenesis provided evidence for the role of N-terminal D109 at the dimeric interface in substrate affinity modulation and thermostabilization. The dimeric Gt-Mamy transglycosylates hydrolytic products of G4/G5 and acarbose, while the truncated form does not because of the lack of extra sugar-binding space formed due to dimerization.Conclusion/significanceN-terminal domain controls enthalpy-driven thermostabilization, substrate-binding affinity and transglycosylation activity of Gt-Mamy by homodimer formation.

Project description:BACKGROUND:?-D-xylosidase is a vital exoglycosidase with the ability to hydrolyze xylooligosaccharides to xylose and to biotransform some saponins by cleaving outer ?-xylose. ?-D-xylosidase is widely used as one of the xylanolytic enzymes in a diverse range of applications, such as fuel, food and the pharmaceutical industry; therefore, more and more studies have focused on the thermostable and xylose-tolerant ?-D-xylosidases. RESULTS:A thermostable ?-xylosidase gene (xln-DT) of 1509 bp was cloned from Dictyoglomus thermophilum and expressed in E.coli BL21. According to the amino acid and phylogeny analyses, the ?-xylosidase Xln-DT is a novel ?-xylosidase of the GH family 39. The recombinant ?-xylosidase was purified, showing unique bands on SDS-PAGE, and had a protein molecular weight of 58.7 kDa. The ?-xylosidase Xln-DT showed an optimal activity at pH 6.0 and 75 °C, with p-nitrophenyl-?-D-xylopyranoside (pNPX) as a substrate. Xln-DT displayed stability over a pH range of 4.0-7.5 for 24 h and displayed thermotolerance below 85 °C. The values of the kinetic parameters K m and V max for pNPX were 1.66 mM and 78.46 U/mg, respectively. In particular, Xln-DT displayed high tolerance to xylose, with 60% activity in the presence of 3 M xylose. Xln-DT showed significant effects on the hydrolyzation of xylobiose. After 3 h, all the xylobiose tested was degraded into xylose. Moreover, ?-xylosidase Xln-DT had a high selectivity for cleaving the outer xylose moieties of natural saponins, such as notoginsenoside R1 and astragaloside IV, which produced the ginsenoside Rg1 with stronger anti-fatigue activity and produced cycloastragenol with stronger anti-aging activity, respectively. CONCLUSION:This study provides a novel GH 39 ?-xylosidase displaying extraordinary properties of highly catalytic activity at temperatures above 75 °C, remarkable hydrolyzing activity of xylooligosaccharides and rare saponins producing ability in the pharmaceutical and commercial industries.

Project description:?-Xylosidase is an important constituent of the hemicellulase system and it plays an important role in hydrolyzing xylooligosaccharides to xylose. Xylose, a useful monose, has been utilized in a wide range of applications such as food, light, chemical as well as energy industry. Therefore, the xylose-tolerant ?-xylosidase with high specific activity for bioconversion of xylooligosaccharides has a great potential in the fields as above.A ?-xylosidase gene (Tth xynB3) of 2,322 bp was cloned from the extremely thermophilic bacterium Thermotoga thermarum DSM 5069 that encodes a protein containing 774 amino acid residues, and was expressed in Escherichia coli BL21 (DE3). The phylogenetic trees of ?-xylosidases were constructed using Neighbor-Joining (NJ) and Maximum-Parsimony (MP) methods. The phylogeny and amino acid analysis indicated that the Tth xynB3 ?-xylosidase was a novel ?-xylosidase of GH3. The optimal activity of the Tth xynB3 ?-xylosidase was obtained at pH 6.0 and 95°C and was stable over a pH range of 5.0-7.5 and exhibited 2 h half-life at 85°C. The kinetic parameters Km and Vmax values for p-nitrophenyl-?-D-xylopyranoside and p-nitrophenyl-?-L-arabinofuranoside were 0.27 mM and 223.3 U/mg, 0.21 mM and 75 U/mg, respectively. The kcat/Km values for p-nitrophenyl-?-D-xylopyranoside and p-nitrophenyl-?-L-arabinofuranoside were 1,173.4 mM-1 s-1 and 505.9 mM-1 s-1, respectively. It displayed high tolerance to xylose, with Ki value approximately 1000 mM. It was stimulated by xylose at higher concentration up to 500 mM, above which the enzyme activity of Tth xynB3 ?-xylosidase was gradually decreased. However, it still remained approximately 50% of its original activity even if the concentration of xylose was as high as 1000 mM. It was also discovered that the Tth xynB3 ?-xylosidase exhibited a high hydrolytic activity on xylooligosaccharides. When 5% substrate was incubated with 0.3 U Tth xynB3 ?-xylosidase in 200 ?L reaction system for 3 h, almost all the substrate was biodegraded into xylose.The article provides a useful and novel ?-xylosidase displaying extraordinary and desirable properties: high xylose tolerance and catalytic activity at temperatures above 75°C, thermally stable and excellent hydrolytic activity on xylooligosaccharides.

Dataset Information

Structural basis of product inhibition by arabinose and xylose of the thermostable GH43 ?-1,4-xylosidase from Geobacillus thermoleovorans IT-08.

Publications

Structural basis of product inhibition by arabinose and xylose of the thermostable GH43 β-1,4-xylosidase from Geobacillus thermoleovorans IT-08.

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