Dataset Information

A new family of carbohydrate esterases is represented by a GDSL hydrolase/acetylxylan esterase from Geobacillus stearothermophilus.

ABSTRACT: Acetylxylan esterases hydrolyze the ester linkages of acetyl groups at positions 2 and/or 3 of the xylose moieties in xylan and play an important role in enhancing the accessibility of xylanases to the xylan backbone. The hemicellulolytic system of the thermophilic bacterium Geobacillus stearothermophilus T-6 comprises a putative acetylxylan esterase gene, axe2. The gene product belongs to the GDSL hydrolase family and does not share sequence homology with any of the carbohydrate esterases in the CAZy Database. The axe2 gene is induced by xylose, and the purified gene product completely deacetylates xylobiose peracetate (fully acetylated) and hydrolyzes the synthetic substrates 2-naphthyl acetate, 4-nitrophenyl acetate, 4-methylumbelliferyl acetate, and phenyl acetate. The pH profiles for k(cat) and k(cat)/K(m) suggest the existence of two ionizable groups affecting the binding of the substrate to the enzyme. Using NMR spectroscopy, the regioselectivity of Axe2 was directly determined with the aid of one-dimensional selective total correlation spectroscopy. Methyl 2,3,4-tri-O-acetyl-?-d-xylopyranoside was rapidly deacetylated at position 2 or at positions 3 and 4 to give either diacetyl or monoacetyl intermediates, respectively; methyl 2,3,4,6-tetra-O-acetyl-?-d-glucopyranoside was initially deacetylated at position 6. In both cases, the complete hydrolysis of the intermediates occurred at a much slower rate, suggesting that the preferred substrate is the peracetate sugar form. Site-directed mutagenesis of Ser-15, His-194, and Asp-191 resulted in complete inactivation of the enzyme, consistent with their role as the catalytic triad. Overall, our results show that Axe2 is a serine acetylxylan esterase representing a new carbohydrate esterase family.

SUBMITTER: Alalouf O

PROVIDER: S-EPMC3234920 | biostudies-literature | 2011 Dec

REPOSITORIES: biostudies-literature

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A new family of carbohydrate esterases is represented by a GDSL hydrolase/acetylxylan esterase from Geobacillus stearothermophilus.

Alalouf Onit O Balazs Yael Y Volkinshtein Margarita M Grimpel Yael Y Shoham Gil G Shoham Yuval Y

The Journal of biological chemistry 20111012 49

Acetylxylan esterases hydrolyze the ester linkages of acetyl groups at positions 2 and/or 3 of the xylose moieties in xylan and play an important role in enhancing the accessibility of xylanases to the xylan backbone. The hemicellulolytic system of the thermophilic bacterium Geobacillus stearothermophilus T-6 comprises a putative acetylxylan esterase gene, axe2. The gene product belongs to the GDSL hydrolase family and does not share sequence homology with any of the carbohydrate esterases in th ...[more]

PMID: 21994937

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Project description:The fungal members of Carbohydrate Esterase family 1 (CE1) from the CAZy database include both acetyl xylan esterases (AXEs) and feruloyl esterases (FAEs). AXEs and FAEs are essential auxiliary enzymes to unlock the full potential of feedstock. They are being used in many biotechnology applications including food and feed, pulp and paper, and biomass valorization. AXEs catalyze the hydrolysis of acetyl group from xylan, while FAEs release ferulic and other hydroxycinnamic acids from xylan and pectin. Previously, we reported a phylogenetic analysis for the fungal members of CE1, establishing five subfamilies (CE1_SF1-SF5). Currently, the characterized AXEs are in the subfamily CE1_SF1, whereas CE1_SF2 contains mainly characterized FAEs. These two subfamilies are more related to each other than to the other subfamilies and are predicted to have evolved from a common ancestor, but target substrates with a different molecular structure. In this study, four ascomycete enzymes from CE1_SF1 and SF2 were heterologously produced in Pichia pastoris and characterized with respect to their biochemical properties and substrate preference toward different model and plant biomass substrates. The selected enzymes from CE1_SF1 only exhibited AXE activity, whereas the one from CE1_SF2 possessed dual FAE/AXE activity. This dual activity enzyme also showed broad substrate specificity toward model substrates for FAE activity and efficiently released both acetic acid and ferulic acid (∼50%) from wheat arabinoxylan and wheat bran which was pre-treated with a commercial xylanase. These fungal AXEs and FAEs also showed promising biochemical properties, e.g., high stability over a wide pH range and retaining more than 80% of their residual activity at pH 6.0-9.0. These newly characterized fungal AXEs and FAEs from CE1 have high potential for biotechnological applications. In particular as an additional ingredient for enzyme cocktails to remove the ester-linked decorations which enables access for the backbone degrading enzymes. Among these novel enzymes, the dual FAE/AXE activity enzyme also supports the evolutionary relationship of CE1_SF1 and SF2.

Project description:GDSL-type esterase/lipase proteins (GELPs) belong to the SGNH hydrolase superfamily and contain a conserved GDSL motif at their N-terminus. GELPs are widely distributed in nature, from microbes to plants, and play crucial roles in growth and development, stress responses and pathogen defense. However, the identification and functional analysis of GELP genes are hardly explored in soybean. This study describes the identification of 194 GELP genes in the soybean genome and their phylogenetic classification into 11 subfamilies (A-K). GmGELP genes are disproportionally distributed on 20 soybean chromosomes. Large-scale WGD/segmental duplication events contribute greatly to the expansion of the soybean GDSL gene family. The Ka/Ks ratios of more than 70% of duplicated gene pairs ranged from 0.1-0.3, indicating that most GmGELP genes were under purifying selection pressure. Gene structure analysis indicate that more than 74% of GmGELP genes are interrupted by 4 introns and composed of 5 exons in their coding regions, and closer homologous genes in the phylogenetic tree often have similar exon-intron organization. Further statistics revealed that approximately 56% of subfamily K members contain more than 4 introns, and about 28% of subfamily I members consist of less than 4 introns. For this reason, the two subfamilies were used to simulate intron gain and loss events, respectively. Furthermore, a new model of intron position distribution was established in current study to explore whether the evolution of multi-gene families resulted from the diversity of gene structure. Finally, RNA-seq data were used to investigate the expression profiles of GmGELP gene under different tissues and multiple abiotic stress treatments. Subsequently, 7 stress-responsive GmGELP genes were selected to verify their expression levels by RT-qPCR, the results were consistent with RNA-seq data. Among 7 GmGELP genes, GmGELP28 was selected for further study owing to clear responses to drought, salt and ABA treatments. Transgenic Arabidopsis thaliana and soybean plants showed drought and salt tolerant phenotype. Overexpression of GmGELP28 resulted in the changes of several physiological indicators, which allowed plants to adapt adverse conditions. In all, GmGELP28 is a potential candidate gene for improving the salinity and drought tolerance of soybean.

Dataset Information

A new family of carbohydrate esterases is represented by a GDSL hydrolase/acetylxylan esterase from Geobacillus stearothermophilus.

Publications

A new family of carbohydrate esterases is represented by a GDSL hydrolase/acetylxylan esterase from Geobacillus stearothermophilus.

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