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

Fig. 5 Overview of chloride depletion workflow for revealing cryptic halogenation^[59]

Extracts from the Article

由于隐秘卤化产物的卤素特征峰在质谱分析中难以识别，传统基于卤素同位素峰或碎片离子的检测方法面临挑战。针对此问题，Glasser等［59］提出一种新型策略：通过监测微生物培养液中卤素离子（如Cl?）的异常消耗，结合非靶向代谢组学筛选潜在卤化产物（图5）。以蓝细菌Nostoc punctiforme ATCC 29133为例，对比其在富Cl?与缺Cl?培养条件下的代谢组，成功鉴定出一类氯化糖脂化合物nostochlorosides。该类分子含古洛糖单元及醚键结构，其生物合成基因簇 ngl 包含两个关键酶编码基因nglC和nglO，分别负责Cl?引入及糖基醚化。进一步实验表明，将卤素消耗信号替换为特定金属离子（如Cu2?）或氨基酸（如酪氨酸）的消耗监测，可扩展此策略用于识别其他修饰类型的前体衍生化合物。这一方法突破了传统卤化产物鉴定的局限性，为隐秘卤化反应及其功能酶的发现提供了高效工具。

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. 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. 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]