PMID:21994937

Citation	Alalouf, O, Balazs, Y, Volkinshtein, M, Grimpel, Y, Shoham, G and Shoham, Y (2011) A new family of carbohydrate esterases is represented by a GDSL hydrolase/acetylxylan esterase from Geobacillus stearothermophilus. J. Biol. Chem. 286:41993-2001
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-β-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-β-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.
Links	PubMed PMC3234920 Online version:10.1074/jbc.M111.301051
Keywords	Acetylesterase/chemistry; Biomass; Carbohydrates/chemistry; Catalysis; Cell Wall/metabolism; Esterases/chemistry; Geobacillus stearothermophilus/metabolism; Kinetics; Magnetic Resonance Spectroscopy/methods; Plants/metabolism; Polysaccharides/chemistry; Renewable Energy; Serine Proteases/chemistry; Substrate Specificity; Xylans/chemistry
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Annotations

Gene product	Qualifier	GO Term	Evidence Code	with/from	Aspect	Extension	Notes	Status
GEOSE:AXE2		GO:0046555: acetylxylan esterase activity	ECO:0000314:		F		Figure 3 shows the deacetylation of xylobiose peracetate by Axe2 at pH 7 visualized on a TLC plate.	complete CACAO 9769
GEOSE:AXE2	enables	GO:0046555: acetylxylan esterase activity	ECO:0000314: direct assay evidence used in manual assertion		F		Seeded From UniProt	complete

References

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