Zheng Yu-Cong, Li Xiaojun, Cha Lide, Paris Jared C, Michael Charalambos, Ushimaru Richiro, Ogasawara Yasushi, Abe Ikuro, Guo Yisong, Chang Wei-Chen
Department of Chemistry, North Carolina State University, Raleigh, North Carolina 27695, United States.
Department of Chemistry, The Carnegie Mellon University, Pittsburgh, Pennsylvania 15213, United States.
J Am Chem Soc. 2025 Feb 19;147(7):6162-6170. doi: 10.1021/jacs.4c17741. Epub 2025 Feb 3.
Despite the diversity of reactions catalyzed by mononuclear iron and 2-oxoglutarate-dependent enzymes, the factors that lead to diverse reaction outcomes beyond canonical hydroxylation remain elusive. Cyclopropanation reactions are of particular interest not only due to the prevalence of cyclopropane moieties in pharmaceuticals but also due to the chemistry that allows cyclopropanation to outcompete oxygen rebound. HrmJ is one such cyclopropanase from the biosynthetic pathway of hormaomycin; however, a homologue is herein discovered that instead catalyzes -hydroxylation of the same nitro enolate substrate. These enzymes were reconstituted with Mn(II) and V(IV)═O as mimics of the resting (Fe(II)) and reactive (Fe(IV)═O) intermediate states, respectively. Corresponding crystal structures of the cyclopropanase bound with a substrate imply H atom transfer via an offline π-pathway. In contrast, analogous structural analysis of the hydroxylase implies H atom abstraction likely proceeds through a σ-pathway. Preparation of isotopically labeled substrates and stopped-flow kinetic analyses indicate that while the hydrogen of C4 is abstracted in both enzymes, the Fe(IV)═O intermediate reacts ca. 17-fold faster in the active site of the hydroxylase, consistent with the mechanistic assignments. These results also support a correlation between the mechanism of H atom transfer and the subsequent fate of the substrate radical once generated. A subtle difference in substrate positioning not only affects the H atom abstraction pathway but also allows the nitro enolate moiety to intercept the resulting substrate radical in the active site of the cyclopropase, thereby facilitating intramolecular C-C bond formation in a stereoselective manner.
尽管单核铁和2-氧代戊二酸依赖性酶催化的反应具有多样性，但导致除经典羟基化之外的多种反应结果的因素仍然难以捉摸。环丙烷化反应特别令人感兴趣，这不仅是因为环丙烷部分在药物中普遍存在，还因为其化学性质使得环丙烷化能够胜过氧反弹。HrmJ是来自霍马霉素生物合成途径的一种这样的环丙烷化酶；然而，本文发现了一种同源物，它反而催化相同硝基烯醇底物的β-羟基化。这些酶分别用Mn(II)和V(IV)═O进行重构，以模拟静止（Fe(II)）和反应性（Fe(IV)═O）中间状态。与底物结合的环丙烷化酶的相应晶体结构表明H原子通过非直接的π-途径转移。相比之下，对羟化酶的类似结构分析表明H原子的夺取可能通过σ-途径进行。同位素标记底物的制备和停流动力学分析表明，虽然两种酶都夺取了C4的氢，但Fe(IV)═O中间体在羟化酶的活性位点反应速度快约17倍，这与机理分配一致。这些结果还支持H原子转移机制与一旦产生的底物自由基的后续命运之间的相关性。底物定位的细微差异不仅影响H原子夺取途径，还使硝基烯醇部分在环丙烷化酶的活性位点拦截产生的底物自由基，从而以立体选择性方式促进分子内C-C键的形成。
