Department of Biological Sciences and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600, Bangi, Selangor, Malaysia.
Department of Chemical Sciences, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600, Bangi, Selangor, Malaysia.
Appl Biochem Biotechnol. 2023 Nov;195(11):6708-6736. doi: 10.1007/s12010-022-04304-w. Epub 2023 Mar 13.
Enzymatic halogenation captures scientific interest considering its feasibility in modifying compounds for chemical diversity. Currently, majority of flavin-dependent halogenases (F-Hals) were reported from bacterial origin, and as far as we know, none from lichenized fungi. Fungi are well-known producers of halogenated compounds, so using available transcriptomic dataset of Dirinaria sp., we mined for putative gene encoding for F-Hal. Phylogenetic-based classification of the F-Hal family suggested a non-tryptophan F-Hals, similar to other fungal F-Hals, which mainly act on aromatic compounds. However, after the putative halogenase gene from Dirinaria sp., dnhal was codon-optimized, cloned, and expressed in Pichia pastoris, the ~63 kDa purified enzyme showed biocatalytic activity towards tryptophan and an aromatic compound methyl haematommate, which gave the tell-tale isotopic pattern of a chlorinated product at m/z 239.0565 and 241.0552; and m/z 243.0074 and 245.0025, respectively. This study is the start of understanding the complexities of lichenized fungal F-hals and its ability to halogenate tryptophan and other aromatic. compounds which can be used as green alternatives for biocatalysis of halogenated compounds.
考虑到其在化合物化学多样性修饰方面的可行性，酶促卤化引起了科学界的兴趣。目前，大多数黄素依赖卤化酶（F-Hal）均来自细菌来源，据我们所知，真菌中尚无此类酶。真菌是卤化化合物的知名生产者，因此，我们利用 Dirinaria sp. 的现有转录组数据集，挖掘可能编码 F-Hal 的基因。基于系统发育的 F-Hal 家族分类表明，非色氨酸 F-Hal 与其他真菌 F-Hal 相似，主要作用于芳香族化合物。然而，在对 Dirinaria sp. 的假定卤化酶基因进行分析后，我们对 dnhal 进行了密码子优化、克隆，并在毕赤酵母中表达，该约 63 kDa 的纯化酶对色氨酸和芳香族化合物甲基血红素表现出生物催化活性，分别在 m/z 239.0565 和 241.0552 以及 m/z 243.0074 和 245.0025 处给出了氯化产物的典型同位素模式。本研究是了解地衣真菌 F-hal 及其卤化色氨酸和其他芳香族化合物的复杂性的开始，这可以作为卤化化合物生物催化的绿色替代物。
