SbzP is a unique pyridoxal-5′-phosphate-dependent enzyme, which catalyses a [3+2] annulation between the pyridinium ring of β-nicotinamide adenine dinucleotide (β-NAD) and an electron rich β,γ-unsaturated quinonoid derived from S-adenosylmethionine in natural product azaindane antibiotics biosynthesis. The SbzP-mediated annulation has been proposed to be a rare tandem C–C bond formation, but its structural basis and catalytic mechanism remain largely unknown. Here we report the β-NAD-complexed structure of PseP (SbzP homologue), identified by cryo-electron microscopy. Structure-based mutagenesis, stopped-flow analysis, thermal shift and surface plasmon resonance analysis identified the important residues for the substrate binding. Molecular dynamics simulations provided insights regarding how the enzyme orients the Cγ of the unsaturated quinonoid to β-NAD. In addition, density functional theory calculations confirmed that the proposed stepwise mechanism is more likely than a pericyclization mechanism. This study provides the structural basis of a pyridoxal-5′-phosphate-dependent enzyme that catalyses nucleophilic Cγ addition and β-NAD processing in natural product biosynthesis.

SbzP 是一种独特的吡哆醛-5′-磷酸依赖性酶，可催化 β-烟酰胺腺嘌呤二核苷酸 (β-NAD) 的吡啶鎓环与源自 β-烟酰胺腺嘌呤二核苷酸 (β-NAD) 的富电子 β,γ-不饱和醌型化合物之间形成 [3+2] 环化。天然产物氮杂茚烷抗生素生物合成中的S-腺苷甲硫氨酸。 SbzP介导的环化被认为是一种罕见的串联C-C键形成，但其结构基础和催化机制仍然很大程度上未知。在这里，我们报告了通过冷冻电子显微镜鉴定的 PseP（SbzP 同源物）的 β-NAD 复合结构。基于结构的诱变、停流分析、热位移和表面等离子体共振分析确定了底物结合的重要残基。分子动力学模拟提供了有关酶如何将不饱和醌类化合物的 Cγ 定向为 β-NAD 的见解。此外，密度泛函理论计算证实，所提出的逐步机制比周环化机制更有可能。这项研究提供了吡哆醛-5′-磷酸依赖性酶的结构基础，该酶可催化天然产物生物合成中的亲核 Cγ 加成和 β-NAD 加工。