A New Family of Carbohydrate Esterases Is Represented by a GDSL Hydrolase/Acetylxylan Esterase from Geobacillus stearothermophilus
A New Family of Carbohydrate Esterases Is Represented by a GDSL Hydrolase/Acetylxylan Esterase from Geobacillus stearothermophilus
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DOI：
10.1074/jbc.m111.301051
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发表时间：
2011-12-09
影响因子：
4.8
通讯作者：
Shoham, Yuval
中科院分区：
文献类型：
--
作者：
Alalouf, Onit;Balazs, Yael;Shoham, Yuval
乙酰木聚糖酯酶水解木聚糖中木糖部分的2和/或3位乙酰基的酯键，并在增强木聚糖酶对木聚糖主链的可及性方面发挥重要作用。嗜热细菌嗜热脂肪地芽孢杆菌T-6的半纤维素分解系统包含推定的乙酰木聚糖酯酶基因axe2。该基因产物属于 GDSL 水解酶家族，与 CAZy 数据库中的任何碳水化合物酯酶不具有序列同源性。 axe2基因由木糖诱导，纯化的基因产物使木二糖全乙酸酯完全脱乙酰化（完全乙酰化）并水解合成底物乙酸2-萘酯、乙酸4-硝基苯酯、乙酸4-甲基伞形酮酯和乙酸苯酯。 k(cat) 和 k(cat)/K-m 的 pH 曲线表明存在两个可电离基团影响底物与酶的结合。使用核磁共振波谱，借助一维选择性全相关波谱直接测定 Axe2 的区域选择性。甲基2,3,4-三-O-乙酰基-β-D-吡喃木糖苷在2位或3位和4位快速脱乙酰基，分别得到二乙酰基或单乙酰基中间体；甲基2,3,4,6-四-O-乙酰基-β-D-吡喃葡萄糖苷最初在6位脱乙酰基。在这两种情况下，中间体的完全水解发生的速度要慢得多，表明首选底物是全乙酸糖形式。 Ser-15、His-194 和 Asp-191 的定点诱变导致酶完全失活，这与它们作为催化三联体的作用一致。总的来说，我们的结果表明 Axe2 是一种丝氨酸乙酰木聚糖酯酶，代表新的碳水化合物酯酶家族。
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-beta-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-beta-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.