Life Sciences Institute and Department of Medicinal Chemistry, University of Michigan, Ann Arbor, Michigan 48109, USA.
Nat Chem. 2011 Jul 17;3(8):628-33. doi: 10.1038/nchem.1087.
Elucidation of natural product biosynthetic pathways provides important insights into the assembly of potent bioactive molecules, and expands access to unique enzymes able to selectively modify complex substrates. Here, we show full reconstitution, in vitro, of an unusual multi-step oxidative cascade for post-assembly-line tailoring of tirandamycin antibiotics. This pathway involves a remarkably versatile and iterative cytochrome P450 monooxygenase (TamI) and a flavin adenine dinucleotide-dependent oxidase (TamL), which act co-dependently through the repeated exchange of substrates. TamI hydroxylates tirandamycin C (TirC) to generate tirandamycin E (TirE), a previously unidentified tirandamycin intermediate. TirE is subsequently oxidized by TamL, giving rise to the ketone of tirandamycin D (TirD), after which a unique exchange back to TamI enables successive epoxidation and hydroxylation to afford, respectively, the final products tirandamycin A (TirA) and tirandamycin B (TirB). Ligand-free, substrate- and product-bound crystal structures of bicovalently flavinylated TamL oxidase reveal a likely mechanism for the C10 oxidation of TirE.
阐明天然产物生物合成途径为研究强效生物活性分子的组装提供了重要的见解，并扩展了获得能够选择性修饰复杂底物的独特酶的途径。在这里，我们展示了 Tirandamycin 抗生素在组装线上进行后修饰的不寻常多步氧化级联的完整体外重建。该途径涉及一种非常灵活和迭代的细胞色素 P450 单加氧酶（TamI）和黄素腺嘌呤二核苷酸依赖性氧化酶（TamL），它们通过反复交换底物共同作用。TamI 将 Tirandamycin C（TirC）羟基化生成 Tirandamycin E（TirE），这是一种以前未被识别的 Tirandamycin 中间体。然后，TirE 被 TamL 氧化，生成 Tirandamycin D（TirD）的酮，然后通过独特的回传到 TamI，分别进行环氧化和羟基化，得到最终产物 Tirandamycin A（TirA）和 Tirandamycin B（TirB）。无配体、底物和产物结合的双共价黄素化 TamL 氧化酶晶体结构揭示了 TirE 的 C10 氧化的可能机制。
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