Structural basis of regioselective tryptophan dibromination by the single-component flavin-dependent halogenase AetF

Abstract

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.

Selective C−H Halogenation of Alkenes and Alkynes Using Flavin‐Dependent Halogenases

“…A recently reported phylogenetic analysis of AetF starting from a much larger number of sequences arrived at a similar set of enzymes, suggesting that relatively few single component FDHs are present in sequence databases. The structures of these sequences predicted using AlphaFold were similar to that of AetF, [47] and importantly, the putative active site lysine residue in these structures overlaid well with the catalytically essential [36] K258 in AetF (Figure S2). The greatest variation in the structures was observed in the C-termini, and analysis of these sequences using TMHMM [48] suggested that this region often contains predicted membrane-spanning regions (see the Supporting Information).…”

“…A) Overlay of models of JamD (yellow) and MCE9 (purple) generated using AlphaFold [50] with the crystal structure of the AetF • Trp • FAD complex (PDB ID 8CJE). [47] B) Effects of E200A, H202A, and Y428F on JamD-catalyzed bromination of phenylacetylene and 1decyne. Conversions determined from relative integration values of starting material and product in GC/MS or LC/MS chromatograms (see the Supporting Information).…”

“…Mutagenesis was used to provide insight into the unique reactivity of JamD and MCE9 toward unactivated alkynes. Models of these enzymes were generated using AlphaFold [50] and overlaid with the crystal structure of the AetF • Trp • FAD complex [47] (Figure 3A). This analysis revealed several residues in the putative active sites of these enzymes that are conserved, including K258, T373, and E200 using AetF residue numbers.…”

Identifying and Engineering Flavin Dependent Halogenases for Selective Biocatalysis

“…17,20,21,63,80,81 Single component FDHs also possess a conserved active site lysine, 2,79 but its distance from the substrate binding site is too great for H-bonding to HOX situated proximal to substrate, suggesting that the role of the lysine and/or the nature of the halogen intermediate may be different in these systems. Our initial modeling incorrectly assumed substrate binding proximal to this lysine in the flavin binding site, 2 but a subsequently published crystal structure showed substrate binding distal to this site 82 . Our recent study showing different responses of single component FDHs to isotope effects also hint at interesting mechanistic complexity to be unraveled.…”

Selective C−H Halogenation of Alkenes and Alkynes Using Flavin‐Dependent Halogenases

“… A) Overlay of models of JamD (yellow) and MCE9 (purple) generated using AlphaFold [50] with the crystal structure of the AetF ⋅ Trp ⋅ FAD complex (PDB ID 8CJE) [47] . B) Effects of E200A, H202A, and Y428F on JamD‐catalyzed bromination of phenylacetylene and 1‐decyne.…”