Uncovering the Origins of Selectivity in Non-Heme Iron Dioxygenase-Catalyzed Tropolone Biosynthesis

Non-heme iron (NHI) enzymes perform diverse oxidative transformations with precise control, which can be challenging to achieve with small molecule catalysts, such as the biosynthesis of tropolone. Among them, Anc3, a reconstructed ancestral α-ketoglutarate (α-KG)-dependent NHI dioxygenase, catalyzes a ring-expansion in fungal tropolone biosynthesis from a cyclohexadienone to afford the tropolone natural product stipitaldehyde (ring-expansion product) alongside 3-hydroxyorcinaldehyde (shunt product). This study reveals how the enzyme environment guides the reaction to the ring-expansion product preferably over the shunt product, where the precise selectivity ratio depends on just a handful of Anc3 residues. In particular, molecular dynamics (MD) and quantum mechanical/molecular mechanical (QM/MM) simulations describe how the substrate binds within the NHI active site and can proceed through two distinct mechanisms, ring-expansion or rebound hydroxylation, to yield the two experimentally observed products. Discovery of a linear relationship of ΔEa values and hydrogen bond distances between Arg191 and the Fe(III)–OH group reveals that inhibition of the rebound hydroxylation step increases selectivity toward ring-expansion. Our findings suggest that the rebound hydroxylation rate is further tuned through the Fe(III)–OH bond strength, as influenced by specific secondary sphere coordination effects around the active site. These influences are largely orthogonal to the ring-expansion mechanism, which is shown to prefer to proceed through a radical pathway. In addition, a cationic pathway initiated by electron transfer from substrate to iron is shown to be unfavorable based upon thermodynamic considerations. Altogether, the atomistic details and reaction mechanisms delineated in this work have the potential to guide the tuning of the reaction pathway in related NHI enzymes for selective oxidation reactions.非血红素铁（Non-heme iron, NHI）酶能够实现精准调控的多样化氧化转化，此类转化往往难以通过小分子催化剂完成，例如托酚酮的生物合成。其中，Anc3是一种经过重构的祖先型α-酮戊二酸（α-ketoglutarate, α-KG）依赖型NHI双加氧酶，它可催化真菌托酚酮生物合成中的环扩张反应：将环己二烯酮转化为托酚酮类天然产物stipitaldehyde（环扩张产物），同时伴随生成3-hydroxyorcinaldehyde（分流产物）。本研究阐明了该酶的微环境如何引导反应优先生成环扩张产物而非分流产物，且其精准的选择性比例仅由少数几个Anc3残基决定。具体而言，分子动力学（Molecular dynamics, MD）与量子力学/分子力学（Quantum mechanical/molecular mechanical, QM/MM）模拟揭示了底物如何结合于NHI酶的活性位点，并可通过两种不同的反应机制——环扩张途径或回弹羟基化途径——生成实验中观测到的两种产物。研究发现，Arg191与Fe(III)–OH基团之间的ΔEa值与氢键距离呈线性相关关系，这表明抑制回弹羟基化步骤可提升对环扩张途径的选择性。我们的研究结果提示，回弹羟基化的反应速率可通过Fe(III)–OH键的键强进一步调控，而该调控效应受活性位点周围特定的二级球配位效应影响。这类调控效应与环扩张机制基本正交，后者被证实更倾向于通过自由基途径进行。此外，基于热力学考量，由底物向铁转移电子引发的阳离子途径被证明是不利的。综上，本研究阐明的原子级细节与反应机制，有望为调控相关NHI酶的反应路径以实现选择性氧化反应提供指导。