SimC7 Is a Novel NAD(P)H-Dependent Ketoreductase Essential for the Antibiotic Activity of the DNA Gyrase Inhibitor Simocyclinone.
SimC7 Is a Novel NAD(P)H-Dependent Ketoreductase Essential for the Antibiotic Activity of the DNA Gyrase Inhibitor Simocyclinone.
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DOI：
10.1016/j.jmb.2015.03.019
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发表时间：
2015-06-19
影响因子：
5.6
通讯作者：
Buttner, Mark J.
中科院分区：
文献类型：
--
作者：
Schaefer, Martin;Le, Tung B. K.;Hearnshaw, Stephen J.;Maxwell, Anthony;Challis, Gregory L.;Wilkinson, Barrie;Buttner, Mark J.
关键词：
Simocyclinone D8 （SD8）是由链霉菌（Streptomyces antibiotic t6040）产生的一种有效的DNA回旋酶抑制剂。对西莫环酮（simmocyclinone, sim）生物合成基因簇进行了测序，并提出了一种假设的生物合成途径。SD8中的四烯连接体被认为是模块化I型聚酮合成酶与两种单功能酶反式作用的产物。其中一种单功能酶SimC7被认为可以弥补聚酮合成酶的两个模块缺失的脱水酶活性。在本研究中，我们报道了SimC7的功能。我们在单个噬菌体人工染色体克隆上分离了整个~ 72kb的sim簇，并在一株为提高抗生素产量而设计的彩色链霉菌中异源产生了simmocyclinone。simC7的缺失导致新的simocyclinone， 7-oxo-SD8的产生，出乎意料地携带一个正常的四烯连接体，但在氨环酮部分发生了改变。我们证明SimC7是一种NAD(P) h依赖的酮还原酶，催化7-oxo-SD8转化为SD8。7-oxo-SD8作为DNA旋转酶抑制剂基本上是无活性的，SimC7对酮基的还原被证明是与酶高亲和力结合的关键。因此，SimC7是一种氨环酮酮还原酶，对氨环酮的生物活性至关重要。在异源系统中表达了~ 75-kb的西莫环酮生物合成簇。SimC7是一种新型的NAD(P) h依赖性酮还原酶。SimC7的功能对DNA旋切酶抑制剂西莫环酮的抗生素活性至关重要。
Simocyclinone D8 (SD8) is a potent DNA gyrase inhibitor produced by Streptomyces antibioticus Tü6040. The simocyclinone (sim) biosynthetic gene cluster has been sequenced and a hypothetical biosynthetic pathway has been proposed. The tetraene linker in SD8 was suggested to be the product of a modular type I polyketide synthase working in trans with two monofunctional enzymes. One of these monofunctional enzymes, SimC7, was proposed to supply a dehydratase activity missing from two modules of the polyketide synthase. In this study, we report the function of SimC7. We isolated the entire ~ 72-kb sim cluster on a single phage artificial chromosome clone and produced simocyclinone heterologously in a Streptomyces coelicolor strain engineered for improved antibiotic production. Deletion of simC7 resulted in the production of a novel simocyclinone, 7-oxo-SD8, which unexpectedly carried a normal tetraene linker but was altered in the angucyclinone moiety. We demonstrate that SimC7 is an NAD(P)H-dependent ketoreductase that catalyzes the conversion of 7-oxo-SD8 into SD8. 7-oxo-SD8 was essentially inactive as a DNA gyrase inhibitor, and the reduction of the keto group by SimC7 was shown to be crucial for high-affinity binding to the enzyme. Thus, SimC7 is an angucyclinone ketoreductase that is essential for the biological activity of simocyclinone. The ~ 75-kb simocyclinone biosynthetic cluster was expressed in a heterologous system. SimC7 is a novel NAD(P)H-dependent ketoreductase. SimC7 function is essential for the antibiotic activity of the DNA gyrase inhibitor simocyclinone.
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