Cloning and characterization of a phosphopantetheinyl transferase from Streptomyces verticillus ATCC15003, the producer of the hybrid peptide-polyketide antitumor drug bleomycin
Cloning and characterization of a phosphopantetheinyl transferase from Streptomyces verticillus ATCC15003, the producer of the hybrid peptide-polyketide antitumor drug bleomycin
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DOI：
10.1016/s1074-5521(01)00047-3
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发表时间：
2001-07-01
影响因子：
--
通讯作者：
Shen, B
中科院分区：
文献类型：
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作者：
Sánchez, C;Du, LC;Shen, B
背景资料：磷酸泛酰巯基乙胺基转移酶（PPTases）通过辅酶A的4 ' -磷酸泛酰巯基乙胺（P-pant）部分与保守的丝氨酸残基的共价连接来催化载体蛋白的翻译后修饰，这是包括脂肪酸、聚酮化合物和非核糖体肽的天然产物的生物合成绝对需要的反应。PPT酶已根据其载体蛋白特异性分类。在含有多个P-pant-需要途径的生物体中，已经表明每个途径具有其自身的PPTase活性。结果：建立了一种利用PCR技术克隆PPTase基因的通用方法，并将其应用于轮枝链霉菌（StreptomycesverticillusATCC 15003）的svp痤疮基因的克隆。轮枝。sip基因被定位到不与任何已知NRPS或PKS簇成簇的独立基因座。Svp蛋白在大肠杆菌中过量生产，纯化至均一，并显示为溶液中的单体。Svp是能够修饰来自S. verticillus或其它链霉菌属物种。与Sfp相比，以前已知的唯一的“混杂的”PPTase，Svp显示出类似的催化效率（k（猫）/K-m）的BlmI PCP，但催化效率增加了346倍的TcmM ACP.Conclusions：PPTases最近被重新分类的结构基础上分为两个亚家族：ACPS-type和Sfp-type。从放线菌中克隆Sfp型PPTases的PCR方法的发展，Sfp型PPTases的识别与次级代谢相关，具有宽松的载体蛋白特异性，以及除了Sfp之外SVP的可用性，应该有助于肽，聚酮化合物，特别是杂合肽-聚酮化合物天然产物的工程生物合成的未来努力。(C)2001爱思唯尔科技有限公司版权所有。
Background: Phosphopantetheinyl transferases (PPTases) catalyze the posttranslational modification of carrier proteins by the covalent attachment of the 4 ' -phosphopantetheine (P-pant) moiety of coenzyme A to a conserved serine residue, a reaction absolutely required for the biosynthesis of natural products including fatty acids, polyketides, and nonribosomal peptides. PPTases have been classified according to their carrier protein specificity. In organisms containing multiple P-pant-requiring pathways, each pathway has been suggested to have its own PPTase activity. However, sequence analysis of the bleomycin biosynthetic gene cluster in Streptomyces verticillus ATCC15003 failed to reveal an associated PPTase gene.Results: A general approach for cloning PPTase genes by PCR was developed and applied to the cloning of the svp acne from S. verticillus. The sip gene is mapped to an independent locus not clustered with any of the known NRPS or PKS clusters. The Svp protein was overproduced in Escherichia coli, purified to homogeneity, and shown to be a monomer in solution. Svp is a PPTase capable of modifying both type I and type II acyl carrier proteins (ACPs) and peptidyl carrier proteins (PCPs) from either S. verticillus or other Streptomyces species. As compared to Sfp, the only 'promiscuous' PPTase known previously, Svp displays a similar catalytic efficiency (k(cat)/K-m) for the BlmI PCP but a 346-fold increase in catalytic efficiency for the TcmM ACP.Conclusions: PPTases have recently been re-classified on a structural basis into two subfamilies: ACPS-type and Sfp-type. The development of a PCR method for cloning Sfp-type PPTases from actinomycetes, the recognition of the Sfp-type PPTases to be associated with secondary metabolism with a relaxed carrier protein specificity, and the availability of Svp, in addition to Sfp, should facilitate future endeavors in engineered biosynthesis of peptide, polyketide, and, in particular, hybrid peptide-polyketide natural products. (C) 2001 Elsevier Science Ltd. All rights reserved.