MODULAR STRUCTURE OF PEPTIDE SYNTHETASES REVEALED BY DISSECTION OF THE MULTIFUNCTIONAL ENZYME GRSA
MODULAR STRUCTURE OF PEPTIDE SYNTHETASES REVEALED BY DISSECTION OF THE MULTIFUNCTIONAL ENZYME GRSA
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DOI：
10.1074/jbc.270.11.6163
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发表时间：
1995-03-17
影响因子：
4.8
通讯作者：
MARAHIEL, MA
中科院分区：
文献类型：
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作者：
STACHELHAUS, T;MARAHIEL, MA
对多肽抗生素非核糖体合成酶一级结构的分析揭示了一个高度保守和有序的结构域结构，这些功能单位长约1000个氨基酸，被认为是氨基酸激活和硫酯形成所必需的。为了描述这个结构域的最小延伸，我们扩增和克隆了GRSA基因的截短片段，编码1098个氨基酸的多功能GRSA1，GRSA1。对过表达的His(6)标记的GRSA衍生物进行亲和纯化，并对缺失突变体的催化活性进行了生化研究，包括依赖三磷酸腺苷的氨基酸活化、羧基硫酸酯的形成以及将共价结合的苯丙氨酸从L-向D-异构体外消旋的能力。这些研究揭示了一个核心片段(PheAT-His)，它包含GRSA的前656个氨基酸残基，恢复了天然蛋白的所有活性，除了苯丙氨酸的外消旋。进一步缺失GRSA核心片段(PheAT-His)C-末端约100个氨基酸，包括可能的硫酯结合基序LGGHSL，产生一个556个氨基酸片段(PHEA-His)，显示苯丙氨酸依赖的氨基酰腺基化，但几乎没有硫酯形成。天然GRSA C末端291个氨基酸的缺失导致外消旋能力的完全丧失(PheATS-His)，但它保留了特定的氨基酸激活和硫酯形成的功能。这一结果从生物化学上定义了多肽合成酶结构域的最小尺寸，并揭示了底物识别和ATP依赖的激活以及硫酯形成和外消旋的功能模块的位置。
Analysis of the primary structure of peptide synthetases involved in non-ribosomal synthesis of peptide antibiotics revealed a highly conserved and ordered domain structure, These functional units, which are about 1000 amino acids in length, are believed to be essential for amino acid activation and thioester formation, To delineate the minimal extension of such a domain, we have amplified and cloned truncated fragments of the grsA gene, encoding the 1098-amino acid multifunctional gramicidin S synthetase 1, GrsA. The overexpressed His(6)-tagged GrsA derivatives were affinity-purified, and the catalytic properties of the deletion mutants were examined by biochemical studies including ATP-dependent amino acid activation, carboxyl thioester formation, and the ability to racemize the covalently bound phenylalanine from L- to the D-isomer, These studies revealed a core fragment (PheAT-His) that comprises the first 656 amino acid residues of GrsA, which restored all activities of the native protein, except racemization of phenylalanine. A further deletion of about 100 amino acids at the C-terminal end of the GrsA core fragment (PheAT-His), including the putative thioester binding motif LGGHSL, produced a 556-amino acid fragment (PheA-His) that shows a phenylalanine-dependent aminoacyl adenylation, but almost no thioester formation. A 291-amino acid deletion at the C terminus of the native GrsA, that contains a putative racemization site resulted in complete loss of racemization ability (PheATS-His), However, it retained the functions of specific amino acid activation and thioester formation, The results presented defined biochemically the minimum size of a peptide synthetase domain and revealed the locations of the functional modules involved in substrate recognition and ATP dependent activation as well as in thioester formation and racemization,