胡萝卜软腐果胶杆菌胡萝卜软腐亚种LY34中编码6-磷酸-β-葡萄糖苷酶的asc操纵子的结构与生化分析
Structural and biochemical analysis of the asc operon encoding 6-phospho-beta-glucosidase in Pectobacterium carotovorum subsp. carotovorum LY34.
作者信息
An Chang Long, Lim Woo Jin, Hong Su Young, Shin Eun Chule, Kim Min Keun, Lee Jong Reoul, Park Sang Ryeol, Woo Jong Gyu, Lim Yong Pyo, Yun Han Dae
机构信息
Division of Applied Life Science, Gyeongsang National University, Chinju 660-701, South Korea.
出版信息
Res Microbiol. 2005 Mar;156(2):145-53. doi: 10.1016/j.resmic.2004.09.010. Epub 2004 Dec 10.
An asc operon of Pectobacterium carotovorum subsp. carotovorum LY34 (Pcc LY34) was isolated from a genomic library in a screen for beta-glucosidase activities. Sequence analysis of the 5618-bp cloned DNA fragment (accession number AY622309) showed three open reading frames (ascG, ascF, and ascB) that are predicted to encode 375, 486, and 476 amino acid proteins, respectively. The AscG ORF shared a high similarity with the Escherichia coli AscG repressor. The AscF ORF shared 81% identity with the E. coli AscF PTS enzyme II(asc), while the AscB ORF was highly similar to 6-phospho-beta-glucosidases and is a member of the glycosyl hydrolase family 1. The purified AscB enzyme hydrolyzed salicin, arbutin, pNPG, and MUG. It exhibited maximal activity at pH 7.0 and 40 degrees C, and its activity was enhanced in the presence of Mg(2+) and Ca(2+). The molecular weight of the enzyme was estimated to be 53 000 Da by SDS-PAGE. Two conserved glutamate residues (Glu(182) and Glu(374)) were shown to be important for AscB activity.
从胡萝卜软腐果胶杆菌胡萝卜软腐亚种LY34（Pcc LY34）的基因组文库中筛选β-葡萄糖苷酶活性时，分离得到了一个asc操纵子。对5618 bp的克隆DNA片段（登录号AY622309）进行序列分析，结果显示有三个开放阅读框（ascG、ascF和ascB），预计分别编码含375、486和476个氨基酸的蛋白质。AscG开放阅读框与大肠杆菌AscG阻遏蛋白具有高度相似性。AscF开放阅读框与大肠杆菌AscF磷酸转移酶II（asc）的一致性为81%，而AscB开放阅读框与6-磷酸-β-葡萄糖苷酶高度相似，是糖基水解酶家族1的成员。纯化后的AscB酶可水解水杨苷、熊果苷、对硝基苯-β-D-葡萄糖苷（pNPG）和4-甲基伞形酮基-β-D-葡萄糖苷（MUG）。它在pH 7.0和40℃时表现出最大活性，在Mg(2+)和Ca(2+)存在时其活性增强。通过SDS-PAGE估计该酶的分子量为53 000 Da。已证明两个保守的谷氨酸残基（Glu(182)和Glu(374)）对AscB活性很重要。
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