The Pseudomonas cellulosa glycoside hydrolase family 51 arabinofuranosidase exhibits wide substrate specificity
The Pseudomonas cellulosa glycoside hydrolase family 51 arabinofuranosidase exhibits wide substrate specificity
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DOI：
10.1042/0264-6021:3580607
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发表时间：
2001-09-15
影响因子：
4.1
通讯作者：
Gilbert, HJ
中科院分区：
文献类型：
--
作者：
Beylot, MH;McKie, VA;Gilbert, HJ
为了研究纤维素假单胞菌从多糖和低聚糖中释放阿拉伯糖的机制，对纤维素假单胞菌基因组DNA文库进行了4-甲基luinbelliferyl- α - l-阿拉伯糖醛酸苷酶（MUAase）活性的筛选。分离到一个MUAase基因（abf51A），该基因编码一个57000 Da的非模块化糖苷水解酶家族（GH） 51 arabinofuranosidase （abf51A）。Abf51A的底物特异性表明，它优先从阿拉伯糖或阿拉伯木聚糖中去除α 1,2-和α 1,3-连接的阿拉伯糖醛酸侧链，并水解α 1,5-连接的阿拉伯寡糖，尽管水解速率要低得多。Abf51A对阿拉伯木聚糖的活性与P. cellulosa基因编码的GH62阿拉伯木聚糖苷酶相似。Abf51A的Glu-194和Glu-321在GH51酶中是保守的，这两个氨基酸分别构成了关键的催化酸/碱和亲核残基。为了验证这一假设，我们对Abf51A突变体E194A和E321A的生化特性进行了评价。这些数据与Glu-194和Glu-321构成Abf51A关键催化残基的观点一致。这些数据与所附论文[Beylot， Emami, McKie, Gilbert and Pell (2001) Biochem]中的结果相结合。[j] . 358, 599-605]，表明P. cellulosa表达一种膜结合的GH51 arabinofuranosidase，该酶在从一系列多糖和低聚糖中释放阿拉伯糖中起关键作用。
To investigate the mechanism by which Pseudomonas cellulosa releases arabinose from polysaccharides and oligosaccharides, a gene library of P. cellulosa genomic DNA was screened for 4-methyluinbelliferyl-alpha -L-arabinofuranosidase (MUAase) activity. A single MUAase gene (abf51A) was isolated, which encoded a non-modular glycoside hydrolase family (GH) 51 arabinofuranosidase (Abf51A) of 57 000 Da. The substrate specificity of the Abf51A showed that it preferentially removed alpha1,2- and alpha1,3-linked arabinofuranose side chains from either arabinan or arabinoxylan, and hydrolysed alpha1,5-linked arabino-oligosaccharides, although at a much lower rate. The activity of Abf51A against arabinoxylan was similar to a GH62 arabinofuranosidase encoded by a P. cellulosa gene. Glu-194 and Glu-321 of Abf51A are conserved in GH51 enzymes, and it has been suggested that these amino acids comprise the key catalytic acid/base and nucleophile residues, respectively. To evaluate this hypothesis the biochemical properties of E194A and E321A mutants of Abf51A were evaluated. The data were consistent with the view that Glu-194 and Glu-321 comprise the key catalytic residues of Abf51A. These data, in conjunction with the results presented in the accompanying paper [Beylot, Emami, McKie, Gilbert and Pell (2001) Biochem. J. 358, 599-605], indicate that P. cellulosa expresses a membrane-bound GH51 arabinofuranosidase that plays a pivotal role in releasing arabinose from a range of polysaccharides and oligosaccharides.