Engineering the Pattern of Protein Glycosylation Modulates the Thermostability of a GH11 Xylanase

Fonseca-Maldonado, Raquel; Vieira, Davi Serradella; Alponti, Juliana Sanchez; Bonneil, Éric; Thibault, Pierre; Ward, Richard J.

doi:10.1074/jbc.m113.485953

Cited by 63 publications

(45 citation statements)

References 45 publications

(49 reference statements)

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“…From our results, Asn-37 is involved in a more homogenous glycosylation and plays a more critical role in enzymatic activity and thermostability. These findings suggest that the glycosylation site modification causes various effects, similar to the previous observation (Fonseca-Maldonado et al 2013). Interestingly, both glycosylation sites are located near to the S/T surface that is supposed to play a role in protein stability of GH11 xylanases.…”

Section: Discussionsupporting

confidence: 74%

Improving the catalytic performance of a GH11 xylanase by rational protein engineering

Cheng

Chen

Huang

et al. 2015

Appl Microbiol Biotechnol

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XynCDBFV from Neocallimastix patriciarum is among the most effective xylanases and holds great potentials in a wide variety of industrial applications. In the present study, several active site residues were modified referring to the instrumental information of the complex structure of XynCDBFV and xylooligosaccharides. Among the 12 single active site mutants, W125F and F163W show increased activity comparing to the wild-type protein. The double mutant W125F/F163W was then generated which displayed nearly 20 % increase in the enzyme activity. Although W125F/F163W showed 5 °C reduction in the optimal temperature, it still preserves similar thermostability and is more active than the wild-type enzyme at temperatures lower than 60 °C. These properties make the double mutant a suitable candidate for commercial applications that involve lower operating temperatures. Furthermore, we investigated the effect of N-glycosylation on the thermostability of XynCDBFV when expressed in the yeast strain Pichia pastoris for industrial use. Two potential glycosylation sites (Asn-37 and Asn-88) were examined, and their roles in enzyme performance were validated. We found that the N-glycosylations of XynCDBFV are related to both catalytic activity and heat stability, with Asn-37 motif playing a dominant role. Collectively, the enzymatic properties of XynCDBFV were improved by molecular engineering, and the influences of N-glycosylations on the enzyme have been clearly elucidated herein.

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Section: Discussionsupporting

confidence: 74%

Improving the catalytic performance of a GH11 xylanase by rational protein engineering

Cheng

Chen

Huang

et al. 2015

Appl Microbiol Biotechnol

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“…While the partial glycosylation slightly affects the thermostability of NfPG5, the huge glycosylation has a marked effect on the thermostability of NfPG4. Dispersed glycosylation sites are suggested to favor stabilizing protein-glycan interactions [32]. Through molecular modeling, we observed that the eight glycosylation sites are dispersed on the protein surface of NfPG4 (data not shown).…”

Section: Effects Of Glycosylation On Enzyme Propertiesmentioning

confidence: 90%

“…Glycosylation is well-known to affect various aspects of enzyme properties [32]. Although the molecular mechanism is poorly understood, the covalently attached glycans can stabilize a protein, increase the resistance of a protein against proteinase degradation, regulate the activity of an enzyme, and help protein folding [32,33].…”

Section: Effects Of Glycosylation On Enzyme Propertiesmentioning

confidence: 99%

Two thermophilic fungal pectinases from Neosartorya fischeri P1: Gene cloning, expression, and biochemical characterization

Meng

Pan

et al. 2015

Journal of Molecular Catalysis B: Enzymatic

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“…Of the over 25 distinct PTMs that have been experimentally identified, phosphorylation, acetylation, and N-linked glycosylation are the most frequent, and extensive databases allow for routine access to statistics and properties of PTMs 118,-110 . It is possible to introduce or remove PTMs into proteins through the modulation of sequence motifs (e.g.…”

Section: Modulating Protein Stabilitymentioning

confidence: 99%

“…In an example of the use of MD in this area, Fonseca-Maldonaldo and Ward investigated the relationship between the pattern of glycosylation and xylanase thermostability by systematically modulating the pattern of glycosylation 118 (Figure 3D). GH11 xylanase from Bacillus subtilis has 6 putative glycosylation sites (N8, N20, N25, N29, N141 and N181) and in WT, 4 sites were glycosylated (N20, N25, N141 and N181) when expressed in Pichia pastoris 118 .…”

Section: Modulating Protein Stabilitymentioning

confidence: 99%

Insights from molecular dynamics simulations for computational protein design

Childers

Daggett

2017

Mol. Syst. Des. Eng.

173

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A grand challenge in the field of structural biology is to design and engineer proteins that exhibit targeted functions. Although much success on this front has been achieved, design success rates remain low, an ever-present reminder of our limited understanding of the relationship between amino acid sequences and the structures they adopt. In addition to experimental techniques and rational design strategies, computational methods have been employed to aid in the design and engineering of proteins. Molecular dynamics (MD) is one such method that simulates the motions of proteins according to classical dynamics. Here, we review how insights into protein dynamics derived from MD simulations have influenced the design of proteins. One of the greatest strengths of MD is its capacity to reveal information beyond what is available in the static structures deposited in the Protein Data Bank. In this regard simulations can be used to directly guide protein design by providing atomistic details of the dynamic molecular interactions contributing to protein stability and function. MD simulations can also be used as a virtual screening tool to rank, select, identify, and assess potential designs. MD is uniquely poised to inform protein design efforts where the application requires realistic models of protein dynamics and atomic level descriptions of the relationship between dynamics and function. Here, we review cases where MD simulations was used to modulate protein stability and protein function by providing information regarding the conformation(s), conformational transitions, interactions, and dynamics that govern stability and function. In addition, we discuss cases where conformations from protein folding/unfolding simulations have been exploited for protein design, yielding novel outcomes that could not be obtained from static structures.

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Engineering the Pattern of Protein Glycosylation Modulates the Thermostability of a GH11 Xylanase

Cited by 63 publications

References 45 publications

Improving the catalytic performance of a GH11 xylanase by rational protein engineering

Improving the catalytic performance of a GH11 xylanase by rational protein engineering

Two thermophilic fungal pectinases from Neosartorya fischeri P1: Gene cloning, expression, and biochemical characterization

Insights from molecular dynamics simulations for computational protein design

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