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

Fig. 10 The proposed catalytic mechanism of BesD by calculation and simulation^[80]

Extracts from the Article

Zhang等［80］系统研究了独立型NHFe/αKGHs家族典型成员BesD的催化机制（图10）。量子力学/分子力学（QM/MM）计算与MD模拟结果表明轴向配位的Fe（IV）=O主导底物C-H键的活化过程。氢原子转移（HAT）导致质子从底物C-H键迁移至Fe（IV）=O中心，伴随底物自由基和Fe（III）-OH中间体的形成。值得注意的是，轴向Fe（III）-OH与底物间的空间位阻显著提高了Cl?配体回弹反应的活化能（ΔG=33.5 kcal/mol）。为克服这一能垒，Fe（III）-OH需经历从轴向到赤道位点的构象重排，该异构化过程有效消除了自由基与金属中心的空间排斥作用（ΔG降至13.3 kcal/mol），使底物自由基得以顺利接近Cl?配体完成卤化反应。此外，SCS内的Asn219残基对Fe（III）-OH的构象转换至关重要。N219A突变体的QM/MM研究表明，当Asn219与琥珀酸的氢键相互作用被破坏时，琥珀酸与Fe（III）-OH呈现双齿配位模式，导致轴向Fe（III）-OH无法完成赤道构象转变（ΔG增加至27.3 kcal/mol）。上述结果强调了SCS影响Fe催化中心构象变化的重要性，也对其他NHFe/αKGHs催化的C-H功能化反应具有启示意义。

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• 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. 8  The proposed catalytic mechanism of PrnA by calculation and simulation^[69]
• Fig. 9  The typical structure of NHFe/αKGHs^[25,70-76]
• 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]