Mechanistic Insights into Dideoxygenation in Gentamicin Biosynthesis
Mechanistic Insights into Dideoxygenation in Gentamicin Biosynthesis
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DOI：
10.1021/acscatal.1c03508
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发表时间：
2021-09-20
期刊：
影响因子：
12.9
通讯作者：
Sun, Yuhui
中科院分区：
文献类型：
--
作者：
Li, Sicong;Bury, Priscila dos Santos;Sun, Yuhui
庆大霉素是一种重要的氨基糖苷类抗生素，用于治疗革兰氏阴性菌引起的感染。虽然庆大霉素的大部分生物合成途径已被阐明，但仍然存在一个有趣的问题，即中间体JI-20 A和JI-20 B如何经历双脱氧形成庆大霉素C复合物。在这里，我们表明，双脱氧过程开始与基因P催化的磷酸化的JI-20 A和JI-20 Ba。磷酸化产物通过两种PLP（吡哆醛5 '-磷酸）依赖性酶的协同作用连续修饰：GenB 3消除水，然后通过GenB 4消除磷酸盐和双键迁移。这些反应中的每一个都释放出亚胺，亚胺水解成酮或醛，然后使用氨基供体通过GenB 3再胺化。重要的是，GenB 3和GenB 4的晶体结构已经指导了定点诱变，以揭示酶功能的关键残基。我们提出了GenB 3和GenB 4的催化机制，这揭示了PLP依赖酶已经无与伦比的催化多功能性。
Gentamicin is an important aminoglycoside antibiotic used for treatment of infections caused by Gram-negative bacteria. Although most of the biosynthetic pathways of gentamicin have been elucidated, a remaining intriguing question is how the intermediates JI-20A and JI-20B undergo a dideoxygenation to form gentamicin C complex. Here we show that the dideoxygenation process starts with GenP-catalyzed phosphorylation of JI-20A and JI-20Ba. The phosphorylated products are successively modified by concerted actions of two PLP (pyridoxal 5'-phosphate)-dependent enzymes: elimination of water and then phosphate by GenB3 and double bond migration by GenB4. Each of these reactions liberates an imine which hydrolyses to a ketone or aldehyde and is then reaminated by GenB3 using an amino donor. Importantly, crystal structures of GenB3 and GenB4 have guided site-directed mutagenesis to reveal crucial residues for the enzymes' functions. We propose catalytic mechanisms for GenB3 and GenB4, which shed light on the already unrivalled catalytic versatility of PLP-dependent enzymes.