Department of Chemistry, Bielefeld University, Universitaetsstrasse 25, 33615 Bielefeld, Germany.
Acta Crystallogr D Struct Biol. 2023 Jul 1;79(Pt 7):596-609. doi: 10.1107/S2059798323004254. Epub 2023 Jun 14.
The flavin-dependent halogenase (FDH) AetF successively brominates tryptophan at C5 and C7 to generate 5,7-dibromotryptophan. In contrast to the well studied two-component tryptophan halogenases, AetF is a single-component flavoprotein monooxygenase. Here, crystal structures of AetF alone and in complex with various substrates are presented, representing the first experimental structures of a single-component FDH. Rotational pseudosymmetry and pseudomerohedral twinning complicated the phasing of one structure. AetF is structurally related to flavin-dependent monooxygenases. It contains two dinucleotide-binding domains for binding the ADP moiety with unusual sequences that deviate from the consensus sequences GXGXXG and GXGXXA. A large domain tightly binds the cofactor flavin adenine dinucleotide (FAD), while the small domain responsible for binding the nicotinamide adenine dinucleotide (NADP) is unoccupied. About half of the protein forms additional structural elements containing the tryptophan binding site. FAD and tryptophan are about 16 Å apart. A tunnel between them presumably allows diffusion of the active halogenating agent hypohalous acid from FAD to the substrate. Tryptophan and 5-bromotryptophan bind to the same site but with a different binding pose. A flip of the indole moiety identically positions C5 of tryptophan and C7 of 5-bromotryptophan next to the tunnel and to catalytic residues, providing a simple explanation for the regioselectivity of the two successive halogenations. AetF can also bind 7-bromotryptophan in the same orientation as tryptophan. This opens the way for the biocatalytic production of differentially dihalogenated tryptophan derivatives. The structural conservation of a catalytic lysine suggests a way to identify novel single-component FDHs.
黄素依赖型卤化酶（FDH）AetF 可依次在色氨酸的 C5 和 C7 位溴化，生成 5,7-二溴色氨酸。与研究充分的二组分色氨酸卤化酶不同，AetF 是一种单组分黄素蛋白单加氧酶。本文呈现了 AetF 单独存在及其与各种底物复合物的晶体结构，这是首次报道单组分 FDH 的实验结构。旋转假对称和拟准二倍体使一个结构的相位确定变得复杂。AetF 在结构上与黄素依赖型单加氧酶相关。它包含两个二核苷酸结合结构域，用于结合 ADP 部分，其序列与 GXGXXG 和 GXGXXA 的共识序列不同。一个大结构域紧密结合辅因子黄素腺嘌呤二核苷酸（FAD），而负责结合烟酰胺腺嘌呤二核苷酸（NADP）的小结构域未被占据。大约一半的蛋白质形成包含色氨酸结合位点的额外结构元件。FAD 和色氨酸相距约 16 Å。它们之间的隧道推测允许活性卤化剂次卤酸从 FAD 扩散到底物。色氨酸和 5-溴色氨酸结合到相同的位点，但结合方式不同。吲哚部分的翻转使色氨酸的 C5 和 5-溴色氨酸的 C7 以相同的位置定位在隧道和催化残基旁边，为两次连续卤化的区域选择性提供了简单的解释。AetF 也可以以与色氨酸相同的取向结合 7-溴色氨酸。这为生物催化生产不同二卤代色氨酸衍生物开辟了道路。催化赖氨酸的结构保守性为鉴定新型单组分 FDH 提供了一种方法。
