CLONING AND EXPRESSION OF THE BETA-N-ACETYLGLUCOSAMINIDASE GENE FROM STREPTOCOCCUS-PNEUMONIAE - GENERATION OF TRUNCATED ENZYMES WITH MODIFIED AGLYCON SPECIFICITY
CLONING AND EXPRESSION OF THE BETA-N-ACETYLGLUCOSAMINIDASE GENE FROM STREPTOCOCCUS-PNEUMONIAE - GENERATION OF TRUNCATED ENZYMES WITH MODIFIED AGLYCON SPECIFICITY
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DOI：
10.1074/jbc.270.15.8805
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发表时间：
1995-04-14
影响因子：
4.8
通讯作者：
BUTTERS, TD
中科院分区：
文献类型：
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作者：
CLARKE, VA;PLATT, N;BUTTERS, TD
通过筛选β - n -乙酰氨基葡萄糖苷酶活性表达文库，获得了肺炎链球菌β - n -乙酰氨基葡萄糖苷酶基因。获得了不同核苷酸大小的克隆，每个克隆都具有芳基糖苷活性，DNA测序显示该基因具有3933个碱基对，具有典型的细菌转录起始和终止序列，并以一个赭色停止密码子终止。计算机分析了1311个氨基酸（144,210 Da）的翻译蛋白，发现了一个序列重复序列，其中包含一个与从细菌到人类等多种物种的其他六种己糖胺酶基因产物同源的序列。还发现了一个氨基端假定的分泌信号肽和一个羧基端细胞分选/锚定基序，这些基序通常在20多种其他革兰氏阳性表面蛋白中发现。在大肠杆菌中表达一个几乎完整的DNA克隆，产生一个功能和真实的β - n -乙酰氨基葡萄糖苷酶，具有与野生型酶相同的糖基特异性。然而，由截断的DNA克隆产生的酶显示出更有限的糖基特异性，并且不能水解含有分割n -乙酰氨基葡萄糖的n -聚糖的末端β 1-2GlcNAc残基。这些克隆的可用性允许对催化和低聚糖识别蛋白结构域进行结构分析，从而增强功能活性。
The gene encoding a beta-N acetylglucosaminidase from Streptococcus pneumoniae has been obtained by screening an expression library for beta-N-acetylglucosaminidase activity. Clones of different nucleotide sizes each having arylglycoside activity were obtained, and DNA sequencing revealed a gene of 3933 base pairs possessing typical bacterial transcription initiation and termination sequences and terminating in an ochre stop codon. Computer analysis of the translated protein of 1311 amino acids (144,210 Da) identified a tandem repeat within which lies a sequence homologous with six other hexosaminidase gene products from a wide variety of species ranging from bacteria to humans. Also found were an amino-terminal putative secretion signal peptide and a carboxyl-terminal cell sorting/anchorage motif typically found in over 20 other Gram-positive surface proteins. The expression of an almost complete DNA clone in Escherichia coli produced a functional and authentic beta-N-acetylglucosaminidase with aglycon specificity identical to the wild-type enzyme. However, enzymes produced from truncated DNA clones show more restricted aglycon specificity and are unable to hydrolyze terminal beta 1-2GlcNAc residues from N-glycans containing a bisecting N-acetylglucosamine, The availability of these clones allows structural analyses to be made of catalytic and oligosaccharide recognition protein domains that enhance functional activity.