Molecular cloning and characterization of a novel β-agarase, AgaB, from marine Pseudoalteromonas sp CY24
Molecular cloning and characterization of a novel β-agarase, AgaB, from marine Pseudoalteromonas sp CY24
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DOI：
10.1074/jbc.m607888200
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发表时间：
2007-02-09
影响因子：
4.8
通讯作者：
Yu, Wengong
中科院分区：
文献类型：
--
作者：
Ma, Cuiping;Lu, Xinzhi;Yu, Wengong
琼脂糖酶通常分为糖苷水解酶家族16、50和86，并且发现其降解琼脂糖以频繁地产生新琼脂二糖、新琼脂四糖或新琼脂六糖作为主要产物。本研究从海洋假交替单胞菌CY 24中克隆了一个新的内切型β-琼胶酶基因agaB。agaB基因编码的新琼胶酶与包括所有糖苷水解酶在内的任何已知蛋白质都没有显著的序列相似性。其降解琼脂糖以产生新琼脂八糖和新琼脂十糖作为主要终产物。基于对不同寡糖的酶动力学和降解模式的分析，琼脂糖酶AgaB似乎具有容纳12个糖单元的大底物结合裂缝，其中朝向还原端的8个糖单元跨越亚位点+1至+8，朝向非还原端的4个糖单元跨越亚位点-4至-1，酶切发生在亚位点-1和+1之间。此外，H-1 NMR分析表明，这种酶水解的糖苷键与异头构型的反转，与其他已知的琼胶酶，是保留。总的来说，AgaB在结构和功能上与其他已知的琼脂糖酶不同，似乎代表了一个新的糖苷水解酶家族。
Agarases are generally classified into glycoside hydrolase families 16, 50, and 86 and are found to degrade agarose to frequently generate neoagarobiose, neoagarotetraose, or neoagarohexaose as the main products. In this study we have cloned a novel endo-type beta-agarase gene, agaB, from marine Pseudoalteromonas sp. CY24. The novel agarase encoded by agaB gene has no significant sequence similarity with any known proteins including all glycoside hydrolases. It degrades agarose to generate neoagarooctaose and neoagarodecaose as the main end products. Based on the analyses of enzymatic kinetics and degradation patterns of different oligosaccharides, the agarase AgaB appears to have a large substrate binding cleft that accommodates 12 sugar units, with 8 sugar units toward the reducing end spanning subsites + 1 to + 8 and 4 sugar units toward the non-reducing end spanning subsites -4 to -1, and enzymatic cleavage taking place between subsites -1 and +1. In addition, H-1 NMR analysis shows that this enzyme hydrolyzes the glycosidic bond with inversion of anomeric configuration, in contrast to other known agarases that are retaining. Altogether, AgaB is structurally and functionally different from other known agarases and appears to represent a new family of glycoside hydrolase.