Dataset Information

The critical role of partially exposed N-terminal valine residue in stabilizing GH10 xylanase from Bacillus sp.NG-27 under poly-extreme conditions.

ABSTRACT: BACKGROUND: Understanding the mechanisms that govern protein stability under poly-extreme conditions continues to be a major challenge. Xylanase (BSX) from Bacillus sp. NG-27, which has a TIM-barrel structure, shows optimum activity at high temperature and alkaline pH, and is resistant to denaturation by SDS and degradation by proteinase K. A comparative circular dichroism analysis was performed on native BSX and a recombinant BSX (R-BSX) with just one additional methionine resulting from the start codon. The results of this analysis revealed the role of the partially exposed N-terminus in the unfolding of BSX in response to an increase in temperature. METHODOLOGY: We investigated the poly-extremophilicity of BSX to deduce the structural features responsible for its stability under one set of conditions, in order to gain information about its stability in other extreme conditions. To systematically address the role of the partially exposed N-terminus in BSX stability, a series of mutants was generated in which the first hydrophobic residue, valine (Val1), was either deleted or substituted with various amino acids. Each mutant was subsequently analyzed for its thermal, SDS and proteinase K stability in comparison to native BSX. CONCLUSIONS: A single conversion of Val1 to glycine (Gly) changed R-BSX from being thermo- and alkali- stable and proteinase K and SDS resistant, to being thermolabile and proteinase K-, alkali- and SDS- sensitive. This result provided insight into the structure-function relationships of BSX under poly-extreme conditions. Molecular, biochemical and structural data revealed that the poly-extremophilicity of BSX is governed by a partially exposed N-terminus through hydrophobic interactions. Such hitherto unidentified N-terminal hydrophobic interactions may play a similar role in other proteins, especially those with TIM-barrel structures. The results of the present study are therefore of major significance for protein folding and protein engineering.

SUBMITTER: Bhardwaj A

PROVIDER: S-EPMC2516601 | biostudies-literature | 2008

REPOSITORIES: biostudies-literature

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The critical role of partially exposed N-terminal valine residue in stabilizing GH10 xylanase from Bacillus sp.NG-27 under poly-extreme conditions.

Bhardwaj Amit A Leelavathi Sadhu S Mazumdar-Leighton Sudeshna S Ghosh Amit A Ramakumar Suryanarayanarao S Reddy Vanga S VS

PloS one 20080826 8

<h4>Background</h4>Understanding the mechanisms that govern protein stability under poly-extreme conditions continues to be a major challenge. Xylanase (BSX) from Bacillus sp. NG-27, which has a TIM-barrel structure, shows optimum activity at high temperature and alkaline pH, and is resistant to denaturation by SDS and degradation by proteinase K. A comparative circular dichroism analysis was performed on native BSX and a recombinant BSX (R-BSX) with just one additional methionine resulting from ...[more]

PMID: 18725971

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Project description:Xylanolytic enzymes have a broad range of applications in industrial biotechnology as biocatalytic components of various processes and products, such as food additives, bakery products, coffee extraction, agricultural silage and functional foods. An increasing market demand has driven the growing interest for the discovery of xylanases with specific industrially relevant characteristics, such as stability at elevated temperatures and in the presence of other denaturing factors, which will facilitate their incorporation into industrial processes. In this work, we report the discovery and biochemical characterization of a new thermostable GH10 xylanase, termed XynDZ5, exhibiting only 26% amino acid sequence identity to the closest characterized xylanolytic enzyme. This new enzyme was discovered in an Icelandic hot spring enrichment culture of a Thermoanaerobacterium species using a recently developed bioinformatic analysis platform. XynDZ5 was produced recombinantly in Escherichia coli, purified and characterized biochemically. This analysis revealed that it acts as an endo-1,4-β-xylanase that performs optimally at 65-75°C and pH 7.5. The enzyme is capable of retaining high levels of catalytic efficiency after several hours of incubation at high temperatures, as well as in the presence of significant concentrations of a range of metal ions and denaturing agents. Interestingly, the XynDZ5 biochemical profile was found to be atypical, as it also exhibits significant exo-activity. Computational modeling of its three-dimensional structure predicted a (β/α)8 TIM barrel fold, which is very frequently encountered among family GH10 enzymes. This modeled structure has provided clues about structural features that may explain aspects of its catalytic performance. Our results suggest that XynDZ5 represents a promising new candidate biocatalyst appropriate for several high-temperature biotechnological applications in the pulp, paper, baking, animal-feed and biofuel industries.

Project description:The functional screening of a Pseudacanthotermes militaris termite gut metagenomic library revealed an array of xylan-degrading enzymes, including P. militaris 25 (Pm25), a multimodular glycoside hydrolase family 10 (GH10). Sequence analysis showed details of the unusual domain organization of this enzyme. It consists of one catalytic domain, which is intercalated by two carbohydrate binding modules (CBMs) from family 4. The genes upstream of the genes encoding Pm25 are susC-susD-unk, suggesting Pm25 is a Xyn10C-like enzyme belonging to a polysaccharide utilization locus. The majority of Xyn10C-like enzymes shared the same interrupted domain architecture and were vastly distributed in different xylan utilization loci found in gut Bacteroidetes, indicating the importance of this enzyme in glycan acquisition for gut microbiota. To understand its unusual multimodularity and the possible role of the CBMs, a detailed characterization of the full-length Pm25 and truncated variants was performed. Results revealed that the GH10 catalytic module is specific toward the hydrolysis of xylan. Ligand binding results indicate that the GH10 module and the CBMs act independently, whereas the tandem CBM4s act synergistically with each other and improve enzymatic activity when assayed on insoluble polysaccharides. In addition, we show that the UNK protein upstream of Pm25 is able to bind arabinoxylan. Altogether, these findings contribute to a better understanding of the potential role of Xyn10C-like proteins in xylan utilization systems of gut bacteria.IMPORTANCE Xylan is the major hemicellulosic polysaccharide in cereals and contributes to the recalcitrance of the plant cell wall toward degradation. Members of the Bacteroidetes, one of the main phyla in rumen and human gut microbiota, have been shown to encode polysaccharide utilization loci dedicated to the degradation of xylan. Here, we present the biochemical characterization of a xylanase encoded by a Bacteroidetes strain isolated from the termite gut metagenome. This xylanase is a multimodular enzyme, the sequence of which is interrupted by the insertion of two CBMs from family 4. Our results show that this enzyme resembles homologues that were shown to be important for xylan degradation in rumen or human diet and show that the CBM insertion in the middle of the sequence seems to be a common feature in xylan utilization systems. This study shed light on our understanding of xylan degradation and plant cell wall deconstruction, which can be applied to several applications in food, feed, and bioeconomy.

Dataset Information

The critical role of partially exposed N-terminal valine residue in stabilizing GH10 xylanase from Bacillus sp.NG-27 under poly-extreme conditions.

Publications

The critical role of partially exposed N-terminal valine residue in stabilizing GH10 xylanase from Bacillus sp.NG-27 under poly-extreme conditions.

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