Active-site modifications of adenylation domains lead to hydrolysis of upstream nonribosomal peptidyl thioester intermediates.
Active-site modifications of adenylation domains lead to hydrolysis of upstream nonribosomal peptidyl thioester intermediates.
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DOI：
10.1021/ja048778y
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发表时间：
2004-04
影响因子：
15
通讯作者：
G. Uguru;Claire Milne;Matthew Borg;F. Flett;Colin P. Smith;Jason Micklefield
中科院分区：
文献类型：
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作者：
G. Uguru;Claire Milne;Matthew Borg;F. Flett;Colin P. Smith;Jason Micklefield
研究了天蓝色链霉菌非核糖体肽合成酶（NRPS）腺苷酸化（A）结构域的定点突变，以构建新型钙依赖抗生素（CDA）。产生CDA NRPS模块7，A-结构域的单点和双点突变体，预测其将该结构域的特异性从Asp改变为Asn。双点突变体产生了一个新的肽CDA2a-7N，其第7位含有Asn。然而，在单点和双点突变体中，CDA-6聚体中间体的显著水解是明显的。对此的一种解释是突变体模块7 A结构域激活Asn而不是Asp;然而，Asn-硫酯中间体仅被上游C结构域受体位点（a）弱识别，从而允许水分子在供体位点（d）中拦截六肽基中间体。
Site-directed mutagenesis of nonribosomal peptide synthetase (NRPS) adenylation (A) domains was investigated as a means to engineer new calcium-dependent antibiotics (CDA) in Streptomyces coelicolor. Single- and double-point mutants of the CDA NRPS module 7, A-domain were generated, which were predicted to alter the specificity of this domain from Asp to Asn. The double-point mutant produced a new peptide CDA2a-7N containing Asn at position 7 as expected. However, in both the single- and the double-point mutants, significant hydrolysis of the CDA-6mer intermediate was evident. One explanation for this is that the mutant module 7 A-domain activates Asn instead of Asp; however, the Asn-thioester intermediate is only weakly recognized by the upstream C-domain acceptor site (a), allowing a water molecule to intercept the hexapeptidyl intermediate in the donor site (d).