Department of Chemistry, Indian Institute of Technology Dharwad, Karnataka 580011, India.
Department of Chemistry, Indian Institute of Technology Dharwad, Karnataka 580011, India.
Bioorg Med Chem Lett. 2023 Oct 1;94:129440. doi: 10.1016/j.bmcl.2023.129440. Epub 2023 Aug 9.
Promysalin is an amphipathic antibiotic isolated from Pseudomonas promysalinigenes (previously Pseudomonas putida RW10S1) which shows potent antibacterial activities against Gram-negative pathogens by inactivating succinate dehydrogenase. Based on the in-vivo studies, promysalin is hypothesized to be assembled from three building blocks: salicylic acid, proline, and myristic acid via a proposed but uncharacterized hybrid NRPS-PKS biosynthetic pathway. So far, no in-vitro biosynthetic studies have been reported for this promising antibiotic. Here, we report the first in-vitro reconstitution and biochemical characterization of two early enzymes on the pathway: PpgH, an isochorismate synthase (IS), and PpgG, an isochorismate pyruvate lyase (IPL) which are involved in the biosynthesis of salicylic acid, the polar fragment of promysalin. We also report a secondary chorismate mutase (CM) activity for PpgG. Based on our biochemical experiments, preliminary mechanistic proposals have been postulated for PpgH and PpgG. We believe this study will lay a strong foundation for elucidating the functions and mechanisms of other intriguing enzymes of the promysalin biosynthesis pathway, which may potentially unravel interesting enzyme chemistries and promote pathway engineering in the future.
普罗密沙林是一种从假单胞菌属（以前称为恶臭假单胞菌 RW10S1）中分离出的两亲性抗生素，它通过使琥珀酸脱氢酶失活显示出对革兰氏阴性病原体的强大抗菌活性。基于体内研究，推测普罗密沙林由水杨酸、脯氨酸和肉豆蔻酸通过拟议但未表征的混合 NRPS-PKS 生物合成途径组装而成。到目前为止，尚未有针对这种有前途的抗生素的体外生物合成研究。在这里，我们报告了该途径上两个早期酶的首次体外重建和生化表征：PpgH，异分支酸合酶（IS）和 PpgG，异分支酸丙酮酸裂解酶（IPL），它们参与普罗密沙林的生物合成，即水杨酸，普罗密沙林的极性片段。我们还报告了 PpgG 的次要分支酸变位酶（CM）活性。基于我们的生化实验，对 PpgH 和 PpgG 提出了初步的机制假设。我们相信这项研究将为阐明普罗密沙林生物合成途径中其他有趣酶的功能和机制奠定坚实的基础，这可能有助于未来揭示有趣的酶化学和促进途径工程。
