The marine microbial natural product salinosporamide A (marizomib) is a potent proteasome inhibitor currently in clinical trials for the treatment of brain cancer. Salinosporamide A is characterized by a complex and densely functionalized γ-lactam-β-lactone bicyclic warhead, the assembly of which has long remained a biosynthetic mystery. Here, we report an enzymatic route to the salinosporamide core catalyzed by a standalone ketosynthase (KS), SalC. Chemoenzymatic synthesis of carrier protein-tethered substrates, as well as intact proteomics, allowed us to probe the reactivity of SalC and understand its role as an intramolecular aldolase/β-lactone synthase with roles in both transacylation and bond-forming reactions. Additionally, we present the 2.85-Å SalC crystal structure that, combined with site-directed mutagenesis, allowed us to propose a bicyclization reaction mechanism. This work challenges our current understanding of the role of KS enzymes and establishes a basis for future efforts toward streamlined production of a clinically relevant chemotherapeutic.

海洋微生物天然产物 salinosporamide A (marizomib) 是一种有效的蛋白酶体抑制剂，目前正在进行治疗脑癌的临床试验。 Salinosporamide A 的特点是具有复杂且密集功能化的 γ-内酰胺-β-内酯双环弹头，其组装长期以来一直是生物合成之谜。在这里，我们报告了由独立的酮合酶（KS）SalC催化的盐孢酰胺核心的酶促途径。载体蛋白连接底物的化学酶合成以及完整的蛋白质组学使我们能够探测 SalC 的反应性并了解其作为分子内醛缩酶/β-内酯合酶在转酰基反应和成键反应中的作用。此外，我们还展示了 2.85-Å SalC 晶体结构，该结构与定点诱变相结合，使我们能够提出双环化反应机制。这项工作挑战了我们目前对 KS 酶作用的理解，并为未来简化临床相关化疗药物的生产奠定了基础。