The Emergence of New Catalytic Abilities in an Endoxylanase from Family GH10 by Removing an Intrinsically Disordered Region (original) (raw)
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Protein Engineering Design and Selection, 2009
Rational protein engineering was applied to improve the limited stability of the glycosyl hydrolase family 11 (GH11) endo-b-1,4-xylanase from Bacillus subtilis under acidic conditions. Since the pH dependence of protein stability is governed by the ionisation states of the side chains of its titrable amino acid residues, we explored the strategy of changing pH-stability profiles by altering pK a values of key residues through in silico designed mutations. To this end, computational predictions and molecular modelling were carried out using the recently developed pKD software package. Four endoxylanase variants, in which the pK a values of either Asp4 and Asp11 or His149 were targeted to shift downwards through incorporation of three to five point mutations, were generated and recombinantly expressed in the cytoplasm of Escherichia coli. All four mutants showed considerably increased functional stability at acid pH levels. They retained 30-70% and 75-95% of their activity after incubation at pH 3 and 4, respectively, in comparison with only 23% and 57%, respectively, for the wild-type enzyme under the experimental conditions. No acidophilic adaptation of the catalytic activity had occurred. In addition, their functional stability and catalytic activity profiles under different temperature and ionic strength conditions were significantly altered. These findings contribute to general understanding of the molecular mechanisms governing the pH-dependent stability of GH11 proteins, and hence they can be applied to enhance the stability and effectiveness of many GH11 endoxylanases used in industry today.
Biotechnology and Bioengineering, 2018
Rational design is an important tool for sculpting functional and stability properties of proteins and its potential can be much magnified when combined with in vitro and natural evolutionary diversity. Herein, we report the structureguided design of a xylose-releasing exo-β-1,4-xylanase from an inactive member of glycoside hydrolase family 43 (GH43). Structural analysis revealed a nonconserved substitution (Lys 247) that results in the disruption of the hydrogen bond network that supports catalysis. The mutation of this residue to a conserved serine restored the catalytic activity and crystal structure elucidation of the mutant confirmed the recovery of the proper orientation of the catalytically-relevant histidine. Interestingly, the tailored enzyme can cleave both xylooligosaccharides and xylan, releasing xylose as the main product, being the first xylose-releasing exo-β-1,4-xylanase reported in the GH43 family. This enzyme presents a unique active-site topology when compared to closely related β-xylosidases, which is the absence of a hydrophobic barrier at the positive-subsite region, allowing the accommodation of long substrates. Therefore, the combination of rational design for catalytic activation along with naturally occurring differences in the substrate binding interface led to the discovery of a novel activity within the GH43 family. In addition, these results demonstrate the importance of solvation of the βpropeller hollow for GH43 catalytic function and expand our mechanistic understanding about the diverse modes of action of GH43 members, a key and This article is protected by copyright. All rights reserved. Accepted Article polyspecific Carbohydrate-Active Enzyme (CAZyme) family abundant in most plant cell-wall-degrading microorganisms.
Substrate Specificity in Glycoside Hydrolase Family 10
Journal of Biological Chemistry, 2000
Endoxylanases are a group of enzymes that hydrolyze the -1,4-linked xylose backbone of xylans. They are predominantly found in two discrete sequence families known as glycoside hydrolase families 10 and 11. The Streptomyces lividans xylanase Xyl10A is a family 10 enzyme, the native structure of which has previously been determined by x-ray crystallography at a 2.
New biotechnology, 2016
Xylanases catalyze the hydrolysis of β-1,4-linked xylosyl moieties from xylan chains, one of the most abundant hemicellulosic polysaccharides found in plant cell walls. These enzymes can exist either as single catalytic domains or as modular proteins composed of one or more carbohydrate-binding modules (CBMs) appended to the catalytic core. However, the molecular mechanisms governing the synergistic effects between catalytic domains and their CBMs are not fully understood. Thus, the goal of this study was to evaluate the functional effects of the fusion of a CBM belonging to family 6, which exhibits high affinity to xylan, with the GH11 xylanase from Bacillus subtilis, which does not have a CBM in its wild-type form. The wild-type enzyme (BsXyl11) and the chimeric protein (BsXyl11-CBM6) were heterologously produced in Escherichia coli and purified to homogeneity for biochemical characterization. The molecular fusion did not alter the pH and temperature dependence, but kinetic data r...
Organic & Biomolecular Chemistry, 2009
Computational simulations have been performed using hybrid quantum-mechanical/ molecular-mechanical potentials to investigate the catalytic mechanism of the retaining endo-b-1, 4-xylanase (BCX) from B. circulans. Two-dimensional potential-of-mean-force calculations based upon molecular dynamics with the AM1/OPLS method for wild-type BCX with a p-nitrophenyl xylobioside substrate in water clearly indicates a stepwise mechanism for glycosylation: the rate-determining step is nucleophilic substitution by Glu78 to form the covalently bonded enzyme-substrate intermediate without protonation of the leaving group by Glu172. The geometrical configuration of the transition state for the enzymic reaction is essentially the same as found for a gas-phase model involving only the substrate and a propionate/propionic acid pair to represent the catalytic glutamate/glutamic acid groups. In addition to stabilizing the 2,5 B boat conformation of the proximal xylose in the non-covalent reactant complex of the substrate with BCX, Tyr69 lowers the free-energy barrier for glycosylation by 42 kJ mol-1 relative to that calculated for the Y69F mutant, which lacks the oxygen atom O Y. B3LYP/6-31+G* energy corrections reduce the absolute height of the barrier to reaction. In the oxacarbenium ion-like transition state O Y approaches closer to the endocyclic oxygen O ring of the sugar ring but donates its hydrogen bond not to O ring but rather to the nucleophilic oxygen of Glu78. Comparison of the average atomic charge distributions for the wild-type and mutant indicates that charge separation along the bond between the anomeric carbon and O ring is matched in the former by a complementary separation of charge along the O Y-H Y bond, corresponding to a pair of roughly antiparallel bond dipoles, which is not present in the latter.
Biochemical and Biophysical Research Communications, 2008
Two combinatorial libraries of glycosyl hydrolase family 11 (GH11) Bacillus subtilis endoxylanase XynA were constructed and displayed on phage. Both phage-displayed libraries were subjected to three consecutive biopanning rounds against immobilized endoxylanase inhibitor TAXI, each time preceded by an incubation step at elevated temperature. DNA sequence analysis of enriched phagemid panning isolates allowed identification of mutations conferring enhanced thermal stability. In particular, substitutions T44C, T44Y, F48C, T87D, and Y94C were retained, and their thermostabilizing effect was confirmed by testing site-directed XynA variants. None of these mutations was identified in earlier endoxylanase engineering studies. Each single mutation increased the half-inactivation temperature by 2-3°C over that of the wild-type enzyme. Intriguingly, the three selected cysteine variants generated dimers by formation of intermolecular disulfide bridges.
Structure Dynamics Guided Enzyme Improvement of ENDO-BETA-1, 4-XYLANASE I
2013
Enzyme structure dynamics has recently been revealed to be essential for structure-function relationship. Among various structure dynamics analysis platforms, hydrogen deuterium exchange mass spectrometry stands as an efficient and highthroughput way to analyze protein dynamics upon ligand binding, protein folding, and enzyme catalysis. HDX-MS can be used to study the regional dynamics of proteins based on the m/z value or percentage of deuterium incorporation for the digested peptides in the HDX experiments. Various software packages have been developed to analyze HDX-MS data. However, for the accurate, enhanced, and explicit statistical analysis of HDX-MS data statistical analysis of software was developed as HDXanalyzer. v ACKNOWLEDGEMENTS I would like to express my deep gratitude to my advisor Dr. Joshua S. Yuan, for his invaluable guidance and suggestions, his constant encouragement and financial support, and his understanding, patience, and friendship in my pursuit of a PhD degree. I
Acidophilic adaptation of family 11 endo-β-1,4-xylanases: Modeling and mutational analysis
Protein Science, 2004
Xyl1 from Streptomyces sp. S38 belongs to the low molecular mass family 11 of endo--1,4-xylanases. Its three-dimensional structure has been solved at 2.0 Å and its optimum temperature and pH for enzymatic activity are 60°C and 6.0, respectively. Aspergillus kawachii xylanase XynC belongs to the same family but is an acidophilic enzyme with an optimum pH of 2.0. Structural comparison of Xyl1 and XynC showed differences in residues surrounding the two glutamic acid side chains involved in the catalysis that could be responsible for the acidophilic adaptation of XynC. Mutations W20Y, N48D, A134E, and Y193W were introduced by site-directed mutagenesis and combined in multiple mutants. Trp 20 and Tyr 193 are involved in substrate binding. The Y193W mutation inactivated Xyl1 whereas W20Y decreased the optimum pH of Xyl1 to 5.0 and slightly increased its specific activity. The N48D mutation also decreased the optimum pH of Xyl1 by one unit. The A134E substitution did not induce any change, but when combined with N48D, a synergistic effect was observed with a 1.4 unit decrease in the optimum pH. Modeling showed that the orientations of residue 193 and of the fully conserved Arg 131 are different in acidophilic and "alkaline" xylanases whereas the introduced Tyr 20 probably modifies the pK a of the acid-base catalyst via residue Asn 48. Docking of a substrate analog in the catalytic site highlighted striking differences between Xyl1 and XynC in substrate binding. Hydrophobicity calculations showed a correlation between acidophilic adaptation and a decreased hydrophobicity around the two glutamic acid side chains involved in catalysis.
Scientific Reports
Enzymes that cleave polysaccharides in lignocellulose, i. e., cellulases, xylanases, and accessory enzymes, play crucial roles in the natural decomposition of plant-derived biomass and its efficient and sustainable processing into biofuels or other bulk chemicals. The analysis of open reading frame cthe_2195 from the thermophilic, cellulolytic anaerobe Clostridium thermocellum (also known as 'Ruminiclostridium thermocellum') suggested that it encoded a cellulosomal protein comprising a dockerin-I module, a carbohydrate-binding module, and a module of previously unknown function. The biochemical characterisation upon recombinant expression in Escherichia coli revealed that the protein is a thermostable endoxylanase, named Xyn141E with an optimal pH of 6.0-6.5 and a temperature optimum of 67-75 °C. The substrate spectrum of Xyn141E resembles that of GH10 xylanases, because of its side activities on carboxymethyl cellulose, barley β-glucan, and mannan. Conversely, the product spectrum of Xyn141E acting on arabinoxylan is similar to those of GH11, as established by HPAEC-PAD analysis. Xyn141E is weakly related (20.7% amino acid sequence identity) to the founding member of the recently established GH family 141 and is the first xylanase in this new family of biomass-degrading enzymes. Research on carbohydrate-degrading enzymes has advanced markedly since their discovery in 1833 1, 2 , with the first descriptions of cellulases in 1912 3 and of xylanases in 1955 4, 5 , as well as the publication of a comprehensive database of carbohydrate-active enzymes (CAZy) in 1998 6. To date, more than 400,000 DNA sequences associated with enzymes targeting carbohydrates have been identified. These have been grouped into five classes: glycoside hydrolases (GH), glycosyl transferases (GT), polysaccharide lyases (PL), carbohydrate esterases (CE), auxiliary activities (AA), and associated carbohydrate-binding modules (CBM). The major class of carbohydrate-degrading enzymes, the GH, includes more than 330,000 enzymes, of which approximately 2% have been characterised. Based on structural similarities, GH enzymes have been grouped into 145 different families 6. Although a vast number of proteins have been characterised and functionally annotated, many putative proteins have not yet been connected to a biological function. The interest in discovering new glycoside hydrolases continues to be high: for example, research on glycoside hydrolases gives insight into fields such as carbohydrate utilisation by human gut microbiota 7 and can provide new functionalities for the efficient breakdown of plant biomass for industrial biotechnology. Further investigation of uncharacterised proteins will contribute to a better understanding and utilisation of carbohydrate-degrading microorganisms, as well as to the annotation of gene function in biological databases. The complex structure of xylan, the second-most abundant polysaccharide 8 , has inspired the evolution of a sophisticated set of enzymes for its degradation. This toolbox includes endo-1,4-β-xylanases (EC 3.2.1.8) for the