Calcium Protects a Mesophilic Xylanase from Proteinase Inactivation and Thermal Unfolding

Spurway, Tracey D.; Morland, Carl; Cooper, Alan; Sumner, Ian G.; Hazlewood, Geoffrey P.; O'Donnell, Anthony G.; Pickersgill, Richard W.; Gilbert, Harry J.

doi:10.1074/jbc.272.28.17523

Cited by 48 publications

(41 citation statements)

References 26 publications

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“…It is located at an equivalent position as the structural calcium ion found in CBM22 (13), bridging the N-and C-terminal regions, although neither the residues that interact with the ion nor the number of different ligation types are identical. Structural calcium ions often play a key role in protein stability (40), and recent data have shown that removal of the divalent ion from CBM6 greatly increased the susceptibility of the protein to proteolytic degradation. 3.…”

Section: Resultsmentioning

confidence: 99%

The Location of the Ligand-binding Site of Carbohydrate-binding Modules That Have Evolved from a Common Sequence Is Not Conserved

Czjzek

Bolam

Mosbah

et al. 2001

Journal of Biological Chemistry

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108

113

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Polysaccharide-degrading enzymes are generally modular proteins that contain non-catalytic carbohydrate-binding modules (CBMs), which potentiate the activity of the catalytic module. CBMs have been grouped into sequence-based families, and three-dimensional structural data are available for half of these families. Clostridium thermocellum xylanase 11A is a modular enzyme that contains a CBM from family 6 (CBM6), for which no structural data are available. We have determined the crystal structure of this module to a resolution of 2.1 Å. The protein is a ␤-sandwich that contains two potential ligand-binding clefts designated cleft A and B. The CBM interacts primarily with xylan, and NMR spectroscopy coupled with site-directed mutagenesis identified cleft A, containing Trp-92, Tyr-34, and Asn-120, as the ligand-binding site. The overall fold of CBM6 is similar to proteins in CBM families 4 and 22, although surprisingly the ligand-binding site in CBM4 and CBM22 is equivalent to cleft B in CBM6. These structural data define a superfamily of CBMs, comprising CBM4, CBM6, and CBM22, and demonstrate that, although CBMs have evolved from a relatively small number of ancestors, the structural elements involved in ligand recognition have been assembled at different locations on the ancestral scaffold.

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Section: Resultsmentioning

confidence: 99%

The Location of the Ligand-binding Site of Carbohydrate-binding Modules That Have Evolved from a Common Sequence Is Not Conserved

Czjzek

Bolam

Mosbah

et al. 2001

Journal of Biological Chemistry

Self Cite

108

113

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“…Enzyme and Stability Assays-The activity of CjXyn10A against 4-methylumbelliferyl-␤-D-cellobioside (MUCase activity) was determined as described previously (6). Xylanase activity was performed essentially as described by Charnock et al (9) except that the release of reducing sugar was determined using the Somogyi-Nelson reagent (22), and the substrate used was 0.2% glucuronoxylan.…”

Section: Bacterial Strains Plasmid Construction and Growth Conditions-mentioning

confidence: 99%

“…The activity of CmXyn10B against 4-nitrophenyl-␤-D-cellobioside was as described by Pell et al (8). Thermo inactivation studies used the methods of Spurway et al (6), and residual activity was assessed by assaying MUCase activity. Differential scanning calorimetry (DSC) and isothermal titration calorimetry were carried out as described previously (6).…”

Section: Bacterial Strains Plasmid Construction and Growth Conditions-mentioning

confidence: 99%

“…CmXyn10B is the most active GH10 xylanase described to date against both xylooligosaccharides and a range of xylans, whereas CjXyn10A displays properties that are typical of extracellular family 10 enzymes (8,9). CjXyn10A displays resistance to proteinase attack and thermal inactivation at temperatures Ͻ65°C in the presence of calcium; however, removal of the divalent metal ion causes a dramatic increase in thermo and proteolytic inactivation (6). Structural and site-directed mutagenesis studies of CjXyn10A show that calcium confers stability by binding to an extended loop (designated loop 7) between ␤-strand 7 and ␣-helix 7 (6).…”

mentioning

confidence: 99%

“…CjXyn10A displays resistance to proteinase attack and thermal inactivation at temperatures Ͻ65°C in the presence of calcium; however, removal of the divalent metal ion causes a dramatic increase in thermo and proteolytic inactivation (6). Structural and site-directed mutagenesis studies of CjXyn10A show that calcium confers stability by binding to an extended loop (designated loop 7) between ␤-strand 7 and ␣-helix 7 (6). The metal ion makes co-ordinating bonds with the OD1 of Asp-262, OD1 and OD2 of Asp-256, OD1 of Asn-261, and the carbonyl groups of Asn-253 and Asn-258, with the octahedral geometry completed by a water molecule.…”

mentioning

confidence: 99%

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The Use of Forced Protein Evolution to Investigate and Improve Stability of Family 10 Xylanases

Andrews

Taylor

Pell

et al. 2004

Journal of Biological Chemistry

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Metal ions such as calcium often play a key role in protein thermostability. The inclusion of metal ions in industrial processes is, however, problematic. Thus, the evolution of enzymes that display enhanced stability, which is not reliant on divalent metals, is an important biotechnological goal. Here we have used forced protein evolution to interrogate whether the stabilizing effect of calcium in an industrially relevant enzyme can be replaced with amino acid substitutions. Our study has focused on the GH10 xylanase CjXyn10A from Cellvibrio japonicus, which contains an extended calcium binding loop that confers proteinase resistance and thermostability. Three rounds of error-prone PCR and selection identified a treble mutant, D262N/A80T/R347C, which in the absence of calcium is more thermostable than wild type CjXyn10A bound to the divalent metal. D262N influences the properties of the calcium binding site, A80T fills a cavity in the enzyme, increasing the number of hydrogen bonds and van der Waals interactions, and the R347C mutation introduces a disulfide bond that decreases the free energy of the unfolded enzyme. A derivative of CjXyn10A (CfCjXyn10A) in which the calcium binding loop has been replaced with a much shorter loop from Cellulomonas fimi CfXyn10A was also subjected to forced protein evolution to select for thermostablizing mutations. Two amino acid substitutions within the introduced loop and the A80T mutation increased the thermostability of the enzyme. This study demonstrates how forced protein evolution can be used to introduce enhanced stability into industrially relevant enzymes while removing calcium as a major stability determinant.

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High resolution structure and sequence ofT. aurantiacus Xylanase I: Implications for the evolution of thermostability in family 10 xylanases and enzymes with ??-barrel architecture

et al. 1999

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Xylanase I is a thermostable xylanase from the fungus Thermoascus aurantiacus, which belongs to family 10 in the current classification of glycosyl hydrolases. We have determined the three-dimensional X-ray structure of this enzyme to near atomic resolution (1.14 A) by molecular replacement, and thereby corrected the chemically determined sequence previously published. Among the five members of family 10 enzymes for which the structure has been determined, Xylanase I from T. aurantiacus and Xylanase Z from C. thermocellum are from thermophilic organisms. A comparison with the three other available structures of the family 10 xylanases from mesophilic organisms suggests that thermostability is effected mainly by improvement of the hydrophobic packing, favorable interactions of charged side chains with the helix dipoles and introduction of prolines at the N-terminus of helices. In contrast to other classes of proteins, there is very little evidence for a contribution of salt bridges to thermostability in the family 10 xylanases from thermophiles. Further analysis of the structures of other proteins from thermophiles with eight-fold (beta)alpha-barrel architecture suggests that favorable interactions of charged side chains with the helix dipoles may be a common way in which thermophilic proteins with this fold are stabilized. As this is the most common type of protein architecture, this finding may provide a useful guide for site-directed mutagenesis aimed to improve the thermostability of (beta)alpha-barrel proteins. Proteins 1999;36:295-306.

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Calcium Protects a Mesophilic Xylanase from Proteinase Inactivation and Thermal Unfolding

Cited by 48 publications

References 26 publications

The Location of the Ligand-binding Site of Carbohydrate-binding Modules That Have Evolved from a Common Sequence Is Not Conserved

The Location of the Ligand-binding Site of Carbohydrate-binding Modules That Have Evolved from a Common Sequence Is Not Conserved

The Use of Forced Protein Evolution to Investigate and Improve Stability of Family 10 Xylanases

High resolution structure and sequence ofT. aurantiacus Xylanase I: Implications for the evolution of thermostability in family 10 xylanases and enzymes with ??-barrel architecture

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