Uguru Gabriel C, Milne Claire, Borg Matthew, Flett Fiona, Smith Colin P, Micklefield Jason
Department of Chemistry, University of Manchester Institute of Science and Technology (UMIST), Manchester, UK.
J Am Chem Soc. 2004 Apr 28;126(16):5032-3. doi: 10.1021/ja048778y.
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).
非核糖体肽合成酶（NRPS）腺苷化（A）结构域的定点诱变被作为一种在天蓝色链霉菌中构建新型钙依赖性抗生素（CDA）的手段进行了研究。生成了CDA NRPS模块7的A结构域的单点和双点突变体，预计这些突变体会将该结构域的特异性从Asp改变为Asn。双点突变体产生了预期在第7位含有Asn的新肽CDA2a-7N。然而，在单点和双点突变体中，CDA-6聚体中间体的显著水解都很明显。对此的一种解释是，突变的模块7 A结构域激活Asn而非Asp；然而，Asn-硫酯中间体仅被上游C结构域接受位点（a）微弱识别，使得水分子能够在供体位点（d）截获六肽基中间体。
