State Key Laboratory of Biocatalysis and Enzyme Engineering, Hubei Hongshan Laboratory, Hubei Collaborative Innovation Center for Green Transformation of Bio-Resources, Hubei Key Laboratory of Industrial Biotechnology, School of Life Sciences, Hubei University, Wuhan, 430062, PR China.
Hubei Gongtong Steroid Drug Research Institute, Wuhan, 430073, PR China.
Nat Commun. 2023 Nov 16;14(1):7425. doi: 10.1038/s41467-023-43285-0.
The biosynthesis of neurotoxin aetokthonotoxin (AETX) that features a unique structure of pentabrominated biindole nitrile involves a first-of-its-kind nitrile synthase termed AetD, an enzyme that shares very low sequence identity to known structures and catalyzes an unprecedented mechanism. In this study, we resolve the crystal structure of AetD in complex with the substrate 5,7-di-Br-L-Trp. AetD adopts the heme oxygenase like fold and forms a hydrophobic cavity within a helical bundle to accommodate the indole moiety. A diiron cluster comprising two irons that serves as a catalytic center binds to the carboxyl O and the amino N of the substrate. Notably, we demonstrate that the AetD-catalyzed reaction is independent of the bromination of the substrate and also solved crystal structures of AetD in complex with 5-Br-L-Trp and L-Trp. Altogether, the present study reveals the substrate-binding pattern and validates the diiron cluster-comprising active center of AetD, which should provide important basis to support the mechanistic investigations into this class of nitrile synthase.
神经毒素 Aetokthonotoxin（AETX）的生物合成具有独特的五溴化双吲哚腈结构，涉及一种首创的腈合酶，称为 AetD，该酶与已知结构的序列同一性非常低，并催化前所未有的机制。在这项研究中，我们解析了 AetD 与底物 5,7-二-Br-L-Trp 复合物的晶体结构。AetD 采用血红素加氧酶样折叠，并在螺旋束内形成一个疏水性腔以容纳吲哚部分。一个由两个充当催化中心的铁组成的双核铁簇与底物的羧基 O 和氨基 N 结合。值得注意的是，我们证明 AetD 催化的反应不依赖于底物的溴化，并且还解决了 AetD 与 5-Br-L-Trp 和 L-Trp 复合物的晶体结构。总之，本研究揭示了底物结合模式，并验证了 AetD 的双核铁簇组成的活性中心，这应该为支持该类腈合酶的机制研究提供重要依据。
