Mori Shogo, Shrestha Sanjib K, Fernández Javier, Álvarez San Millán María, Garzan Atefeh, Al-Mestarihi Ahmad H, Lombó Felipe, Garneau-Tsodikova Sylvie
Department of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky , Lexington, Kentucky 40536-0596, United States.
Departamento de Biología Funcional e Instituto Universitario de Oncología del Principado de Asturias, Universidad de Oviedo , Oviedo 33006, Spain.
Biochemistry. 2017 Aug 29;56(34):4457-4467. doi: 10.1021/acs.biochem.7b00661. Epub 2017 Aug 17.
The initiation of the nonribosomal peptide synthetase (NRPS) assembly of the bisintercalator natural product thiocoraline involves key enzymatic steps for AMP activation and carrier protein loading of the starter unit 3-hydroxyquinaldic acid (3HQA). Gene cluster data combined with protein sequence homology analysis originally led us to propose that TioJ could be responsible for the AMP activation step, whereas TioO could act as the thiolation (T) domain, facilitating the transfer of 3HQA to the next NRPS module, TioR. Herein, we confirmed the involvement of TioJ in thiocoraline biosynthesis by tioJ knockout and in vitro activation of 3HQA studies. However, we demonstrated that TioJ-activated 3HQA is not loaded onto the T domain TioO, as originally believed, but instead onto a fatty acid synthase (FAS) acyl carrier protein (ACP) domain FabC, which is located outside of the thiocoraline gene cluster. We showed a strong interaction between TioJ and FabC. By generating TioJ point mutants mimicking the active site of highly homologous enzymes activating different molecules, we showed that the identity of the substrate activated by adenylation domains such as TioJ is not determined by only the active site residues that directly interact with the substrate. The insights gained from these enzymatic transformations are valuable in the efforts toward deciphering the complete biosynthetic pathway of thiocoraline and bisintercalators in general.
双嵌入剂天然产物硫珊瑚素的非核糖体肽合成酶（NRPS）组装起始过程涉及起始单元3-羟基喹哪啶酸（3HQA）的AMP激活和载体蛋白装载的关键酶促步骤。基因簇数据与蛋白质序列同源性分析最初使我们提出，TioJ可能负责AMP激活步骤，而TioO可能作为硫醇化（T）结构域，促进3HQA向下一个NRPS模块TioR的转移。在此，我们通过敲除tioJ和3HQA的体外激活研究证实了TioJ参与硫珊瑚素的生物合成。然而，我们证明，TioJ激活的3HQA并非如最初所认为的那样装载到T结构域TioO上，而是装载到位于硫珊瑚素基因簇之外的脂肪酸合酶（FAS）酰基载体蛋白（ACP）结构域FabC上。我们展示了TioJ与FabC之间的强相互作用。通过生成模拟激活不同分子的高度同源酶活性位点的TioJ点突变体，我们表明，诸如TioJ等腺苷化结构域激活的底物的身份并非仅由直接与底物相互作用的活性位点残基决定。从这些酶促转化中获得的见解对于全面解析硫珊瑚素和双嵌入剂的完整生物合成途径具有重要价值。
