Key Laboratory of Industrial Fermentation Microbiology, College of Bioengineering, Tianjin University of Science & Technology, Tianjin, 300457, PR China.
Key Laboratory of Industrial Fermentation Microbiology, College of Bioengineering, Tianjin University of Science & Technology, Tianjin, 300457, PR China.
Eur J Med Chem. 2020 Oct 15;204:112639. doi: 10.1016/j.ejmech.2020.112639. Epub 2020 Jul 19.
Ficellomycin is an aziridine-containing antibiotic, produced by Streptomyces ficellus. Based on the newly identified ficellomycin gene cluster and the assigned functions of its genes, a possible pathway for aziridine ring formation in ficellomycin was proposed, which is a complex process involving at least 3 enzymatic steps. To obtain support for the proposed mechanism, the targeted genes encoding sulfate adenylyltransferase, adenylsulfate kinase, and a putative sulfotransferase were respectively disrupted and the subsequent analysis of their fermentation products revealed that all the three genes were involved in aziridine formation. To further confirm the mechanism, the key gene encoding a putative sulfotransferase was over expressed in Escherichia coli Rosseta (DE3). Enzyme assays indicated that the expressed sulfotransferase could specifically transfer a sulfo group from 3'-phosphoadenosine-5'-phosphosulfate (PAPS) onto the hydroxyl group of (R)-(-)-2-pyrrolidinemethanol. This introduces a good leaving group in the form of the sulfated hydroxyl moiety, which is then converted into an aziridine ring through an intramolecular nucleophilic attack by the adjacent secondary amine. The sulfation/intramolecular cyclization reaction sequence maybe a general strategy for aziridine biosynthesis in microorganisms. Discovery of this mechanism revealed an enzyme-catalyzed route for the synthesis of aziridine-containing reagents and provided an important insight into the functional diversity of sulfotransferases.
菲来霉素是一种含氮杂环的抗生素，由灰色链霉菌产生。根据新鉴定的菲来霉素基因簇及其基因的功能，提出了菲来霉素中环形成的可能途径，这是一个涉及至少 3 个酶促步骤的复杂过程。为了获得对所提出机制的支持，分别敲除了编码硫酸腺苷酰转移酶、腺苷酸硫酸激酶和一个假定的磺基转移酶的靶基因，随后对其发酵产物的分析表明，这三个基因都参与了氮杂环的形成。为了进一步证实这一机制，在大肠杆菌罗斯塔（DE3）中过表达了编码假定磺基转移酶的关键基因。酶促反应表明，表达的磺基转移酶可以特异性地将 3'-磷酸腺苷-5'-磷酸硫酸（PAPS）上的磺基基团转移到（R）-（-）-2-吡咯烷甲醇的羟基上。这引入了一个良好的离去基团，形式为磺化的羟基部分，然后通过相邻仲胺的分子内亲核攻击转化为氮杂环。磺化/分子内环化反应序列可能是微生物中环氮杂环丙烷生物合成的一般策略。这一机制的发现揭示了一种酶催化的合成含氮杂环试剂的方法，并为磺基转移酶的功能多样性提供了重要的见解。
