Structural basis of the substrate subsite and the highly thermal stability of xylanase 10B from <i>Thermotoga maritima</i> MSB8

Ihsanawati,; Kumasaka, Takashi; Kaneko, Tomonori; Morokuma, Chihiro; Yatsunami, Rie; Sato, Takao; Nakamura, Satoshi; Tanaka, Nobuo

doi:10.1002/prot.20700

Cited by 63 publications

(53 citation statements)

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“…Currently, xylanase has been attracting much attention due to its wide biotechnological applications in the food, animal feed, pulp and paper, and textile industries and in the deconstruction of lignocellulose for biofuel production (2)(3)(4)(5)(6). On the basis of the catalytic domains, xylanases are mainly classified into glycoside hydrolase (GH) families 5,8,10,11,30, and 43 (7); GH10 and GH11 xylanases are the best studied.…”

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confidence: 99%

“…To obtain thermostable xylanases, one strategy is to search for novel xylanases from extremophiles. A number of thermostable GH10 xylanases have been reported for thermophilic Thermotoga maritima MSB8 (90°C) (10), Thermotoga petrophila RKU-1 (90°C) (11), and Thermobifida alba UL JB1 (80°C) (12) and for acidomesophilic Bispora sp. strain MEY-1 (85°C) (13).…”

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confidence: 99%

“…strain MEY-1 (85°C) (13). The available sequences and structures of these thermophilic xylanases (i.e., 1VBU and 3NIY [10,11]) provide valuable information for better understanding of the structural and functional characters of thermostable enzymes.…”

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Thermostability Improvement of a Streptomyces Xylanase by Introducing Proline and Glutamic Acid Residues

Wang

Luo

Tian

et al. 2014

Appl Environ Microbiol

100

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cProtein engineering is commonly used to improve the robustness of enzymes for activity and stability at high temperatures. In this study, we identified four residues expected to affect the thermostability of Streptomyces sp. strain S9 xylanase XynAS9 through multiple-sequence analysis (MSA) and molecular dynamic simulations (MDS). Site-directed mutagenesis was employed to construct five mutants by replacing these residues with proline or glutamic acid (V81P, G82E, V81P/G82E, D185P/S186E, and V81P/G82E/ D185P/S186E), and the mutant and wild-type enzymes were expressed in Pichia pastoris. Compared to the wild-type XynAS9, all five mutant enzymes showed improved thermal properties. The activity and stability assays, including circular dichroism and differential scanning calorimetry, showed that the mutations at positions 81 and 82 increased the thermal performance more than the mutations at positions 185 and 186. The mutants with combined substitutions (V81P/G82E and V81P/G82E/D185P/ S186E) showed the most pronounced shifts in temperature optima, about 17°C upward, and their half-lives for thermal inactivation at 70°C and melting temperatures were increased by >9 times and approximately 7.0°C, respectively. The mutation combination of V81P and G82E in adjacent positions more than doubled the effect of single mutations. Both mutation regions were at the end of long secondary-structure elements and probably rigidified the local structure. MDS indicated that a long loop region after positions 81 and 82 located at the end of the inner ␤-barrel was prone to unfold. The rigidified main chain and filling of a groove by the mutations on the bottom of the active site canyon may stabilize the mutants and thus improve their thermostability.

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Thermostability Improvement of a Streptomyces Xylanase by Introducing Proline and Glutamic Acid Residues

Wang

Luo

Tian

et al. 2014

Appl Environ Microbiol

100

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“…In several reports, the structure of the thermostable family 10 xylanase was solved and the implications for the evolution of thermostability in family 10 xylanases and other (α β)8 barrel enzymes were discussed. 29,30) The structural analysis of family 10 xylanases outlined in the papers suggests that thermostability is acquired mainly by improved hydrophobic packing, 29,30) favorable interactions of charged sidechains with helix dipoles, 29,30) and the introduction of proline residues at the N terminus of helices. 29) However, the authors do not refer to the most terminal helices of the enzyme (e.g.…”

Section: Discussionmentioning

confidence: 99%

Importance of Interactions of the .ALPHA.-Helices in the Catalytic Domain N- and C-Terminals of the Family 10 Xylanase from Streptomyces olivaceoviridis E-86 to the Stability of the Enzyme

金子¹,

伊藤²,

藤本³

et al. 2009

J. Appl. Glycosci.

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The plant cell wall consists mainly of a complex mixture of polysaccharides such as cellulose, hemicellulose and pectin. 1) Xylan is the major component of the hemicelluloses found in plant cell walls. The backbone of xylan is formed by β 1,4 linked D xylopyranose units to which several side groups such as α 1,2 linked 4 O methyl D glucuronic acid and α 1,3 linked L arabinofuranose are attached. 2)β Xylanase (EC 3.2.1.8) randomly hydrolyzes β 1,4 glycosidic linkages within the xylan backbone to yield short chain xylooligosaccharides of varying length. Xylanases have many commercial uses, such as in the paper manufacturing, animal feed, bread making, juice and wine industries, and xylitol and xylooligosaccharide production.3)Thermostability is an important property of industrially significant enzymes. Understanding the structural basis for this attribute will underpin the future biotechnological exploitation of these biocatalysis. However, the term thermostability does not refer to a well defined physico chemical property of a protein, but is generally used to indicate the ability of some proteins to function at elevated temperatures and to withstand them for some time. Such an ability implies that the protein is able to remain folded in its functional native state under such conditions. Because many other factors besides temperature affect protein stability, thermostability at best can only be loosely defined. A number of previous studies have shown that various factors can increase protein thermostability, such as an efficient packing of the hydrophobic core, 4 6) electrostatic interactions such as salt bridges and hydrogen bonds, 5,7,8) and the stabilization of helix dipoles. 9)Streptomyces olivaceoviridis E 86 produces both family 10 and 11 xylanases. 10,11) The family 10 xylanase (SoXyn10A) has been well characterized.10,12 22) SoXyn10A consists of two domains, a catalytic domain (belonging to GH family 10) and a carbohydrate binding module (CBM) (belonging to CBM family 13), both of which are connected by a Gly Pro rich linker region.10) The deletion of the CBM of SoXyn10A resulted in about a 20 C decrease in the optimum temperature of SoXyn10A from Streptomyces olivaceoviridis E 86 after removal of the C terminal CBM. Abstract: The intact crystal structure of family 10 xylanase (SoXyn10A) from Streptomyces olivaceoviridis indicates that the catalytic domain of SoXyn10A consists of nine α-helices (α0 8) and eight β-sheets (β1 8). Interaction in the α-helices of N-terminal (α0) and C-terminal (α8) of catalytic domain of SoXyn10A by 3 hydrogen bonds and 8 hydrophobic interactions is observed and predicted to playing an important role for the stability of the molecule. Therefore, the importance for the stability and folding of SoXyn10A were examined by using C-terminal truncated mutants of SoXyn10A. The thermostability was gradually decreased when the C-terminal was shortened; however, the enzyme activities were not influenced by the length of the C-terminal. The investigation of the stability using guanidine ...

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“…Structural conservation and rigidity are demonstrated by the small RMSD (< 3 A) between crystalline and solution structures [12,13]. Furthermore, in-depth intra-family structural comparisons vary by approximately 1 A (RMSD) upon superimposition of the C a of the catalytic domains, despite significant differences in enzyme thermostabilities, and whether or not the xylanase was complexed with xylobiose or related compounds [14].…”

Section: Introductionmentioning

confidence: 99%

Identifying critical unrecognized sugar–protein interactions in GH10 xylanases from Geobacillus stearothermophilus using STD NMR

et al. 2013

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1H solution NMR spectroscopy is used synergistically with 3D crystallographic structures to map experimentally significant hydrophobic interactions upon substrate binding in solution under thermodynamic equilibrium. Using saturation transfer difference spectroscopy (STD NMR), a comparison is made between wild‐type xylanase XT6 and its acid/base catalytic mutant E159Q – a non‐active, single‐heteroatom alteration that has been previously utilized to measure binding thermodynamics across a series of xylooligosaccharide–xylanase complexes [Zolotnitsky et al. (2004) Proc Natl Acad Sci USA 101, 11275–11280). In this study, performing STD NMR of one substrate screens binding interactions to two proteins, avoiding many disadvantages inherent to the technique and clearly revealing subtle changes in binding induced upon mutation of the catalytic Glu. To visualize and compare the binding epitopes of xylobiose–xylanase complexes, a ‘SASSY’ plot (saturation difference transfer spectroscopy) is used. Two extraordinarily strong, but previously unrecognized, non‐covalent interactions with H2–5 of xylobiose were observed in the wild‐type enzyme but not in the E159Q mutant. Based on the crystal structure, these interactions were assigned to tryptophan residues at the −1 subsite. The mutant selectively binds only the β–xylobiose anomer. The 1H solution NMR spectrum of a xylotriose–E159Q complex displays non‐uniform broadening of the NMR signals. Differential broadening provides a unique subsite assignment tool based on structural knowledge of face‐to‐face stacking with a conserved tyrosine residue at the +1 subsite. The results obtained herein by substrate‐observed NMR spectroscopy are discussed further in terms of methodological contributions and mechanistic understanding of substrate‐binding adjustments upon a charge change in the E159Q construct.

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Structural basis of the substrate subsite and the highly thermal stability of xylanase 10B from Thermotoga maritima MSB8

Cited by 63 publications

References 60 publications

Thermostability Improvement of a Streptomyces Xylanase by Introducing Proline and Glutamic Acid Residues

Thermostability Improvement of a Streptomyces Xylanase by Introducing Proline and Glutamic Acid Residues

Importance of Interactions of the .ALPHA.-Helices in the Catalytic Domain N- and C-Terminals of the Family 10 Xylanase from Streptomyces olivaceoviridis E-86 to the Stability of the Enzyme

Identifying critical unrecognized sugar–protein interactions in GH10 xylanases from Geobacillus stearothermophilus using STD NMR

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