A novel arylesterase active toward 7-aminocephalosporanic acid from Agrobacterium radiobacter IFO 12607: Nucleotide sequence, gene expression in Escherichia coli, and site-directed mutagenesis

Yasuyoshi Sakai , Kaoru Ayukawa , Ryoji Mitsui , Hiroya Yurimoto , Keizo Yamamoto , Nobuo Kato
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引用次数: 2

Abstract

A novel arylesterase from Agrobacterium radiobacter IFO 12607 catalyzes the deacetylation of 7-aminocephalosporanic acid (7-ACA) to form deacetyl 7-ACA, but is inactive with cephalosporin C. A DNA fragment carrying the gene encoding the 7-ACA-deacetylating enzyme was cloned from the chromosomal DNA of this bacterium. The open reading frame encoding the enzyme was 642 bp long, corresponding to a protein of 214 amino acid residues (molecular mass=23,085). The deduced amino acid sequence did not contain the sequence GXSXG, typical of the many serine esterases including Bacillus cephalosporin C deacetylase, but has the pentapeptide motif sequence GDSLT (amino acid position 9–13) which is also a consensus sequence of some serine esterases. The newly cloned gene was expressed in Escherichia coli under the control of the lac promoter, and the gene product purified from E. coli exhibited the same catalytic properties as the enzyme purified from A. radiobacter. Site-directed mutagenesis of S11A or S11C within the pentapeptide motif sequence led to complete loss of the enzyme activity. Thus, the Ser-11 residue within the GDSLT motif sequence was determined to construct the catalytic center. These results together with those of our previous studies indicated that the 7-ACA-deacetylating enzyme from A. radiobacter IFO 12607 is a new member of the family of lipolytic serine esterases containing the GDSLT sequence as their catalytic center.

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放射农杆菌IFO 12607对7-氨基头孢酸具有活性的新型芳基酯酶:核苷酸序列、基因在大肠杆菌中的表达和位点定向诱变
从放射农杆菌IFO 12607中分离出一种新的芳基酯酶,该酶能催化7-氨基头孢素酸(7-ACA)的去乙酰化,生成去乙酰化的7-ACA,但对头孢菌素c无活性。编码该酶的开放阅读框长642 bp,对应214个氨基酸残基的蛋白(分子质量=23,085)。推导出的氨基酸序列不包含包括芽孢杆菌C脱乙酰酶在内的许多丝氨酸酯酶的典型序列GXSXG,而是包含五肽基序GDSLT(氨基酸位置9-13),这也是一些丝氨酸酯酶的共识序列。新克隆的基因在lac启动子的控制下在大肠杆菌中表达,从大肠杆菌中纯化的基因产物与从放射杆菌中纯化的酶具有相同的催化性能。S11A或S11C在五肽基序序列内的定点突变导致酶活性完全丧失。因此,确定GDSLT基序序列内的Ser-11残基构建催化中心。这些结果与我们之前的研究结果一起表明,放射杆菌IFO 12607的7- aca -去乙酰化酶是含有GDSLT序列作为催化中心的脂溶丝氨酸酯酶家族的新成员。
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