A flavin-dependent halogenase from metagenomic analysis prefers bromination over chlorination

Flavin-dependent halogenases catalyse halogenation of aromatic compounds. In most cases, this reaction proceeds with high regioselectivity and requires only the presence of FADH2, oxygen, and halide salts. Since marine habitats contain high concentrations of halides, organisms populating the oceans might be valuable sources of yet undiscovered halogenases. A new Hidden-Markov-Model (HMM) based on the PFAM tryptophan halogenase model was used for the analysis of marine metagenomes. Eleven metagenomes were screened leading to the identification of 254 complete or partial putative flavin-dependent halogenase genes. One predicted halogenase gene (brvH) was selected, codon optimised for E. coli, and overexpressed. Substrate screening revealed that this enzyme represents an active flavin-dependent halogenase able to convert indole to 3-bromoindole. Remarkably, bromination prevails also in a large excess of chloride. The BrvH crystal structure is very similar to that of tryptophan halogenases but reveals a substrate binding site that is open to the solvent instead of being covered by a loop.黄素依赖性卤化酶（Flavin-dependent halogenases）可催化芳香族化合物的卤化反应。在多数情况下，该反应具有高度区域选择性，仅需黄素腺嘌呤二核苷酸还原态（FADH2）、氧气与卤化物盐即可进行。由于海洋生境中卤化物浓度较高，海洋栖息生物或许是尚未被发掘的卤化酶的宝贵资源库。本研究基于PFAM色氨酸卤化酶模型构建了全新的隐马尔可夫模型（Hidden-Markov-Model, HMM），用于海洋宏基因组分析。研究对11个宏基因组样本进行筛选，最终鉴定出254条完整或部分的推定黄素依赖性卤化酶基因。研究选取其中一条预测的卤化酶基因（brvH），将其密码子优化以适配大肠杆菌（E. coli）并实现过表达。底物筛选实验结果显示，该酶属于具有活性的黄素依赖性卤化酶，可将吲哚转化为3-溴吲哚。值得注意的是，即便在氯化物过量的条件下，溴化反应仍占据主导地位。BrvH的晶体结构与色氨酸卤化酶的晶体结构极为相似，但其底物结合位点暴露于溶剂环境中，而非被环结构所覆盖。