Synergism between genome sequencing, tandem mass spectrometry and bio-inspired synthesis reveals insights into nocardioazine B biogenesis
Synergism between genome sequencing, tandem mass spectrometry and bio-inspired synthesis reveals insights into nocardioazine B biogenesis
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DOI：
10.1039/c5ob00537j
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发表时间：
2015-01-01
影响因子：
3.2
通讯作者：
Viswanathan, Rajesh
中科院分区：
文献类型：
--
作者：
Alqahtani, Norah;Porwal, Suheel K.;Viswanathan, Rajesh
海洋放线菌衍生的天然产物继续激发化学和生物学的研究。Nocardioazines A和B（3和4）来自Nocardiopsis sp. cmm - m0232，是结构独特的生物碱，具有2,5-二酮哌嗪（DKP）核心，被吲哚C3-戊烯基以及吲哚C3-和n -甲基功能化。他们集会的逻辑仍然是神秘的。Nocardiopsis sp. CMB-M0232草图基因组的生物信息学分析发现noz簇横跨基因组的两个区域，编码在nocardioazine生物合成中起作用的开放阅读框，包括环二肽合成酶（CDPS）、戊烯基转移酶、甲基转移酶和细胞色素P450同源物。来自noz簇的12个基因的异源表达导致环- l - trp - l - trp DKP的积累(5)。通过实验将该簇与吲哚类生物碱天然产物的生物合成联系起来。noz途径的生物信息学分析结果以及放线菌遗传学的挑战促使我们使用不对称合成和质谱技术来确定noz途径中的生物合成中间体。假设的生物合成中间体5和12-17的结构通过化学合成得到了确定。LC-MS和ms将这些合成化合物与Nocardiopsis sp. CMB-M0232化学提取物中的代谢物进行比较，揭示了这些假设的中间体中哪些与nocardioazine生物合成途径有关。这建立了生物合成途径的早期和中期阶段，表明诺卡多毛菌在诺卡多嗪B组装中进行吲哚c3 -甲基化先于吲哚c3 -正常戊酰化和吲哚N1'-甲基化。这些结果强调了融合生物信息学分析、不对称合成方法和质谱代谢物分析在探测天然产物生物合成中的效用。
Marine actinomycete-derived natural products continue to inspire chemical and biological investigations. Nocardioazines A and B (3 and 4), from Nocardiopsis sp. CMB-M0232, are structurally unique alkaloids featuring a 2,5-diketopiperazine (DKP) core functionalized with indole C3-prenyl as well as indole C3- and N-methyl groups. The logic of their assembly remains cryptic. Bioinformatics analyses of the Nocardiopsis sp. CMB-M0232 draft genome afforded the noz cluster, split across two regions of the genome, and encoding putative open reading frames with roles in nocardioazine biosynthesis, including cyclodipeptide synthase (CDPS), prenyltransferase, methyltransferase, and cytochrome P450 homologs. Heterologous expression of a twelve gene contig from the noz cluster in Streptomyces coelicolor resulted in accumulation of cyclo-L-Trp-L-Trp DKP (5). This experimentally connected the noz cluster to indole alkaloid natural product biosynthesis. Results from bioinformatics analyses of the noz pathway along with challenges in actinomycete genetics prompted us to use asymmetric synthesis and mass spectrometry to determine biosynthetic intermediates in the noz pathway. The structures of hypothesized biosynthetic intermediates 5 and 12-17 were firmly established through chemical synthesis. LC-MS and MS-MS comparison of these synthetic compounds with metabolites present in chemical extracts from Nocardiopsis sp. CMB-M0232 revealed which of these hypothesized intermediates were relevant in the nocardioazine biosynthetic pathway. This established the early and mid-stages of the biosynthetic pathway, demonstrating that Nocardiopsis performs indole C3-methylation prior to indole C3-normal prenylation and indole N1'-methylation in nocardioazine B assembly. These results highlight the utility of merging bioinformatics analyses, asymmetric synthetic approaches, and mass spectrometric metabolite profiling in probing natural product biosynthesis.