Dissecting the low catalytic capability of flavin-dependent halogenases

Abstract

Although flavin-dependent halogenases (FDHs) are attractive biocatalysts, their practical applications are limited because of their low catalytic efficiency. Here, we investigated the reaction mechanisms and structures of tryptophan 6-halogenase (Thal) from Streptomyces albogriseolus using stopped-flow, rapid-quench flow, quantum/mechanics molecular mechanics calculations, crystallography, and detection of intermediate (hypohalous acid [HOX]) liberation. We found that the key flavin intermediate, C4a-hydroperoxyflavin (C4aOOH-FAD), formed by Thal and other FDHs (tryptophan 7-halogenase [PrnA] and tryptophan 5-halogenase [PyrH]), can react with I^-, Br^-, and Cl^- but not F^- to form C4a-hydroxyflavin and HOX. Our experiments revealed that I^- reacts with C4aOOH-FAD the fastest with the lowest energy barrier and have shown for the first time that a significant amount of the HOX formed leaks out as free HOX. This leakage is probably a major cause of low product coupling ratios in all FDHs. Site-saturation mutagenesis of Lys79 showed that changing Lys79 to any other amino acid resulted in an inactive enzyme. However, the levels of liberated HOX of these variants are all similar, implying that Lys79 probably does not form a chloramine or bromamine intermediate as previously proposed. Computational calculations revealed that Lys79 has an abnormally lower pKa compared with other Lys residues, implying that the catalytic Lys may act as a proton donor in catalysis. Analysis of new X-ray structures of Thal also explains why premixing of FDHs with reduced flavin adenine dinucleotide generally results in abolishment of C4aOOH-FAD formation. These findings reveal the hidden factors restricting FDHs capability which should be useful for future development of FDHs applications.

Keywords: QM/MM calculations; X-ray structures; flavin monooxygenase; halogenase; kinetics; stopped-flow.

Figure 1

Reaction of Thal with tryptophan. Thal catalyzes halogenation of tryptophan to yield halogenated tryptophan as the product. FAD, oxidized flavin adenine dinucleotide; FADH⁻, reduced flavin adenine dinucleotide.

Figure 2

Kinetic traces of the Thal reaction in the presence of halide ions. An air-saturated solution of Thal premixed with NaCl (blue lines) or NaBr (green lines) was mixed with an anaerobic solution of FADH⁻. Black lines are kinetic traces without addition of halide ions. Flavin intermediates were monitored by absorption changes at 380 nm (solid lines) and 450 nm (dashed lines). C4aOOH-FAD, C4a-hydroperoxyflavin; FADH⁻, reduced flavin adenine dinucleotide.

Figure 3

Kinetic mechanisms of the Thal reaction with tryptophan. FAD refers to oxidized FAD. FADH⁻ refers to reduced FAD. Thal:FADH^-∗ refers to inactive complex which cannot form C4aOOH-FAD. TRP refers to tryptophan. C4aOOH-FAD refers to C4a-hydroperoxyflavin. C4aOH-FAD refers to C4a-hydroxyflavin. X⁻ represents the halide ions. HOX represents hypohalous acid. In this model, TRP can bind to Thal together with a flavin.

Figure 4

Kinetic traces of the Thal reaction with various halide ions. Formation of C4aOH-FAD was monitored at Em > 495 nm using excitation wavelengths of 380 nm (solid lines) and 450 nm (solid lines with circles) in the presence of various halide ions (Cl⁻ (blue), Br⁻ (orange), I⁻ (green), and F⁻ (red)). Gray lines are reactions without addition of halide ion. Note that the difference in fluorescence intensities was because of the difference of photomultiplier tube voltage used for the various halide reactions. Inset shows a plot of the observed rate constants of C4aOH-FAD formation resulting from the reaction of C4aOOH-FAD with halide ions (Cl⁻ (circle), Br⁻ (square), and I⁻ (triangular)) at various concentrations. C4aOOH-FAD, C4a-hydroperoxyflavin; C4aOH-FAD, C4a-hydroxyflavin; Em, emission wavelength.

Figure 5

Detection of HOBr leakage from Thal (wild-type). HPLC-MS/MS chromatograms of the reaction without Thal (black) and the reaction in the presence of Thal (red). Multiple turnover reactions were performed to generate HOBr. A reaction solution after removal of enzymes was incubated with D-luciferin. Reactions were analyzed using HPLC-MS/MS. Mass spectra of bromo-D-luciferin and dibromo-D-luciferin were analyzed using HPLC-QTOF (inset). HOBr, hypobromous acid; QTOF, Quadrupole Time-of-Flight.

Figure 6

Kinetics of Lys79Thr reacting with oxygen. An air-saturated solution of Lys79Thr (blue lines) or wild-type (black lines) (30 μM) was mixed with a solution of FADH⁻ (15 μM) using a single mixing stopped-flow spectrometer. Kinetic traces were monitored at 380 nm (solid lines) and 450 nm (dashed lines). FADH⁻, reduced flavin adenine dinucleotide.

Figure 7

ComparingD-luciferin bromination acitivity of Lys79Thr with wild-type Thal. Peak areas of brominated D-luciferin product of Lys79Thr (orange), wild-type (green), and the reaction without Thal (blue).

Figure 8

Proposed reaction mechanisms of tryptophan halogenation. The reaction was proposed to occur via electrophilic aromatic substitution. A, in tryptophan 7-halogenase (RebH), the Walsh group proposed that tryptophan can be halogenated by a covalent intermediate (Lys-NH-X) (27). B, alternatively, HOX can directly interact with a fully protonated Lys via hydrogen bonding (29, 32) to halogenate tryptophan. X represents I, Br, and Cl. Our findings support mechanism B and suggest the role of Lys in protonation of HOX. HOX, hypohalous acid.

Figure 9

Kinetics of Thal:FADH^-∗ inactive complex formation. An anaerobic solution of FADH⁻ was premixed with an anaerobic solution of Thal at various age times (incubation times) (0.01–30 s) in the first mixing. Aerobic buffer was then added into the second mixing step. Final concentrations were 128 μM oxygen, 15 μM FADH⁻, and 30 μM Thal. Kinetic traces were monitored at 380 nm (solid lines) and 450 nm (solid lines with circles). The inset was a plot of absorbance decrease at 380 nm versus incubation time. FADH⁻, reduced flavin adenine dinucleotide.

Figure 10

Alignment of various conformations of the flavin binding loop of Thal structures. Superposition of conformations of Chain A (green) and Chain B (white) observed in the Thal:FADH⁻ complex (PDB code 7CU2) defined as “loose” and “tight” conformations, respectively, and Chain A (blue) of Thal:FAD:AMP (PDB code 7CU1) defined as “open” conformation. FAD, oxidized flavin adenine dinucleotide; FADH⁻, reduced flavin adenine dinucleotide.

Figure 11

Water networks observed in the open conformation of Chain A of Thal:FAD:AMP (PDB code 7CU1).A, residues within 4 Å of water networks (green sphere). B, spherical display of water molecules shows water networks connecting bulk solvent to the FAD binding site. FAD, oxidized flavin adenine dinucleotide.

Figure 12

Rearrangement of water networks resulting from protein dynamic changes.A, water network arrangements around the flavin site in Chain A of the Thal:FAD:AMP complex (PDB code 7CU1) were analyzed by MD simulations. Arrangement of water networks observed in the structure of the Thal:FAD:AMP complex (PDB code 7CU1). B, rearrangement of water molecules and the green loop after 4 ns of the production phase performed by MD simulation. C, close-up views of interactions of water molecules around the isoalloxazine ring after 4 ns of the production phase by MD simulations. MD, molecular dynamics; FAD, oxidized flavin adenine dinucleotide.

Figure 13

Mechanism of inactive complex formation in FDHs.A, water networks located around the flavin were identified in the crystal structure of Thal. Preincubation of Thal with FADH⁻ under anaerobic conditions promotes conformational changes resulting in water rearrangement around the flavin. These water molecules may promote proton transfer to N1 of FADH⁻ to produce FADH₂ which is less reactive with O₂ to form C4aOOH-FAD (inactive complex). B, freshly mixing Thal with FADH⁻ under aerobic conditions can avoid inactive complex formation. C4aOOH-FAD, C4a-hydroperoxyflavin; FAD, oxidized flavin adenine dinucleotide; FDHs, flavin-dependent halogenases; FADH⁻, reduced flavin adenine dinucleotide; HOX, hypohalous acid.

Figure 14

Comparison of structures of Thal (PDB code 7CU0) and VirX1 (PDB code 6QGM).A, alignment of VirX1 (purple) and Thal (gray) structures identified the distinct α-helical (yellow) structure encompassing the Trp substrate (green stick) in Thal. B, surface representation of VirX1 aligned with Thal.