Halogenases in Biocatalysis: Advances in Mechanism Elucidation， Directed Evolution， and Green Manufacturing Synthetic Biology Journal    DOI: 10.12211/2096-8280.2024-091

Fig. 8 The proposed catalytic mechanism of PrnA by calculation and simulation^[69]

Extracts from the Article

FDHs的催化机制一直存在争议。已有实验和理论研究分别支持HOX对底物的直接卤化［27］（机制1）或在底物卤化之前形成Lys-NH₂Cl+（或Lys-NHCl）中间体［26］（机制2）。最近，Barker等［69］以色氨酸卤化酶PrnA为研究对象，利用密度泛函理论（DFT）和分子动力学（MD）模拟探讨了这两种机制。研究者从HOCl与色氨酸在酶活性位点的结合稳定性入手，最终计算并比较了机制1和机制2的能量分布。通过计算模拟活性位点Lys79和Glu346的全部四种质子化状态，表明状态A（质子化的Lys79和去质子化的Glu346）对于HOCl稳定结合于活性位点是必需的，也是这两种机制催化过程中最可能的一种质子化状态。DFT计算显示，质子化状态A也是机制1的优选状态，因为Lys79与HOCl之间的强氢键增强了HOCl的反应性，使其能够对由HOCl解离形成的羟基进行质子化，同时Glu346的羧酸根则稳定形成的Wheland中间体并随即对其去质子化（图8）。此外，四种质子化状态下，机制2的能量屏障均显著高于机制1，因此可以排除机制2。这些计算为FDHs中HOX对底物直接卤化而非形成赖氨酸中间体的催化机制提供了证据，并揭示了Lys79和Glu346这两个关键残基在初始结合底物及卤化反应过程中的双重作用。

Other Images/Table from this Article

• Fig. 1  Halogens are the most common and important modification groups in pesticides and pharmaceutical products
• Fig. 2  Examples of halogenated natural products and the role of halogenases in molecular synthesis^[4,12,15-16]
• Fig. 3  Cofactors and catalytic mechanisms of different types of halogenase^[1,4,17-25]
• Fig. 4  Workflow of genome mining for halogenases and their application examples^[34]
• Table 1  Examples of halogenase discovered by genome mining
• Table 2  The uniqueness and superiority of the CLEAN algorithm framework
• Fig. 5  Overview of chloride depletion workflow for revealing cryptic halogenation^[59]
• Fig. 6  The typical structure and catalytic elements of flavin-dependent halogenases
• Fig. 7  The typical structure of single-component flavin-dependent halogenases
• Fig. 9  The typical structure of NHFe/αKGHs^[25,70-76]
• Fig. 10  The proposed catalytic mechanism of BesD by calculation and simulation^[80]
• Fig. 11  The action mode and engineering strategies of novel nucleotide halogenase ^[46]
• Fig.12  The structural, function and catalytic mechanism of copper-dependent halogenase ApnU^[81]
• Table 3  Typical engineering cases of natural halogenase in recent years
• Fig. 13  The typical structure SAM-dependent halogenase ^[84-91]The red lines indicate the residues that are crucial for fluorination activity; the yellow lines indicate the residues that can form hydrogen bonds with the modification groups at the C2′ position of adenine
• Fig. 14  Tunnel engineering improves the catalytic performance of Thal halogenase^[96]
• Fig. 15  Directed evolution enhances the fluorination activity of non-heme iron oxidase^[108-109]
• Fig. 16  Biocatalytic synthesis platform of halogenated tryptophan derivatives^[113]
• Tab. 4  Examples of FDHs catalyzing enantioselective reactions^[125-127]
• Tab. 5  Recent cases and key data related to the potential industrial application of halogenases
• Fig. 17  Representative research progress in the field of halogenase research
• Fig. 18  AI/ML drives the intelligent evolution of halogenase^[138-156]