Structural Basis for Methine Excision by a Heme Oxygenase-like Enzyme

Abstract

Heme oxygenase-like domain-containing oxidases (HDOs) are a rapidly expanding enzyme family that typically use dinuclear metal cofactors instead of heme. FlcD, an HDO from the opportunistic pathogen Pseudomonas aeruginosa, catalyzes the excision of an oxime carbon in the biosynthesis of the copper-containing antibiotic fluopsin C. We show that FlcD is a dioxygenase that catalyzes a four-electron oxidation. Crystal structures of FlcD reveal a mononuclear iron in the active site, which is coordinated by two histidines, one glutamate, and the oxime of the substrate. Enzyme activity, Mössbauer spectroscopy, and electron paramagnetic resonance spectroscopy analyses support the usage of a mononuclear iron cofactor. This cofactor resembles that of mononuclear non-heme iron-dependent enzymes and breaks the paradigm of dinuclear HDO cofactors. This study begins to illuminate the catalytic mechanism of methine excision and indicates convergent evolution of different lineages of mononuclear iron-dependent enzymes.

Figure 1

The sequence and structure of the carbon-excision enzyme FlcD. (A) Biosynthesis of fluopsin C. FlcD catalyzes the excision of the oxime carbon as formate, which originates from carbon-2 of l-cysteine (yellow dot). Carbons 1–3 are labeled. SAM, S-adenosyl methionine. (B) Sequence alignment of FlcD and FlcE to other characterized HDOs. The conserved metal-binding residues (aspartate/glutamate in red and histidine in blue) are located on three core helices (α1, α2, and α3). The last two metal ligands of typical HDOs are replaced by V278 and R281 in FlcD (yellow highlight). (C) Homodimeric structure of FlcD with bound Fe (orange) and substrate (green) in two views, one that shows both the N-terminal and the HDO domains (top) and the other with 90° rotation that focuses on the HDO domain (bottom). Monomer A (light blue/black) and monomer B (blue/light gray) each contain a single iron in the HDO domain. The core α-helices are labeled.

Figure 2

FlcD contains a single iron in each monomer and undergoes a conformational change upon substrate binding. (A) Structure of monomer C of FlcD bound to Fe (FlcD·Fe, PDB:9B9M), showing six residues that align with the metal-binding motif in other HDOs (yellow sticks), Fe (orange sphere), and α3 helix (bright yellow). This structure contains four monomers (two dimers) in the asymmetric unit. Monomers A, B, and D exhibit similar structures to monomer C. (B) Structure of monomer A of FlcD bound to Fe and substrate (FlcD·Fe·substrate, PDB: 9B9N), showing six residues that align with the metal-binding motif in other HDOs (light and dark blue sticks), substrate (green), Fe (orange), and the α3 helix (dark blue). (C) Overlayed structures of the α3 helix of the FlcD·Fe monomer C (yellow) and FlcD·Fe·substrate monomer A (blue). Fe (orange). The loop containing V278 and R281 forms a continuous α3 helix upon substrate binding, resulting in an ∼10 Å movement of the Cα of R281. (D–F) The active site of (D) FlcD·Fe monomer C, (E) FlcD·Fe·substrate monomer A, and (F) FlcD·Fe·substrate monomer B. Gray dashed boxes in (D–F) highlight the movement of R281 upon substrate binding. Side chain of R281 is not resolved in (D). Omit map of substrate is shown at 1.0 σ (green mesh). Fo-Fc map (red mesh) of substrate and iron anomalous signal (black mesh) are shown at 3.0 σ. (G, H) Active site of FlcD·Fe·substrate (G) monomer A and (H) monomer B. Key residues (sticks) include iron-binding and substrate-binding residues. Polar, metal–ligand, and charge–charge interactions are shown (gray dashes). Carbons 2 and 3 are labeled. Fe (orange), waters (red), substrate (green).

Figure 3

RFQ Mössbauer and EPR spectroscopic characterization of the reaction of FlcD·Fe(II)·substrate with O₂ supports a mononuclear iron cofactor. (A) 80 K Mössbauer spectra of an anoxic solution of FlcD (1.7 mM) reconstituted with 1 molar equiv of Fe(II) and 3 molar equiv of substrate, as is or reacted with an O₂-saturated buffer (1.8 mM) at 5 °C and quenched at selected time points. The spectra were acquired in the absence of an external magnetic field. The experimental spectra are shown as black vertical bars, the quadrupole doublets corresponding to the fit of the Fe(II) component are shown as red solid lines, and the isomer shift (dark blue) and ΔE_Q (green) are listed in each spectrum. Over time, a small amount of Fe(III) accumulates (blue trace) and its spectrum is obtained after subtraction of the ferrous quadrupole doublet fit from the experimental spectrum. (B) 4.2 K Mössbauer spectra of the same samples as in (A) and in the presence of a small external magnetic field (0.078 T) applied parallel to the direction of the γ-beam. (C) 4.2 K Mössbauer spectra of an anoxic solution of FlcD (1.7 mM) containing 1 molar equiv of Fe(II), 3 molar equiv of substrate, and 10 μM chlorite dismutase (Cld) reacted with a buffer solution containing 7.5 mM sodium chlorite. The experimental spectra are shown as black vertical bars; the quadrupole doublets corresponding to the fit of the Fe(II) component are shown as red solid lines. The asterisks in the 300 s spectrum highlight the positions of the mononuclear high-spin Fe(III) that accumulates in the reaction. (D) Time-dependent product formation in the reactions of FlcD reconstituted with 1 (red) or 2 (blue) molar equiv of Fe(II). Conditions are identical to those of the RFQ Mössbauer experiment with the exception of the Fe(II) molar equiv. Reactions were quenched by the addition of an equal volume of 3.5% H₂SO₄ (chemical quench). Product formation was quantified based on the absorbance peak at 250 nm from LC analysis. Product identity was confirmed using HRMS. E) CW normal mode EPR spectrum of FlcD (1.7 mM) reconstituted with 1 molar equiv of Fe(II) and 3 molar equiv of substrate reacted with an O₂-saturated buffer (1.8 mM) at 5 °C for 300 s, after subtraction of the anaerobic control to remove any background signals due to adventitious high-spin Fe(III) (g ∼ 4.3). The inset contains time-dependent raw spectra centered on the low-field region that were obtained after reaction with O₂ at time points 0 ms (red), 10 ms (blue), 45 s (yellow), and 300 s (black), respectively. The asterisk indicates mononuclear Fe(III). Experimental conditions: T = 10 K, microwave frequency 9.36 GHz, microwave power 2 mW, and modulation amplitude 1 mT.

Figure 4

Oxygen and deuterium isotope tracing experiments demonstrate that FlcD is a dioxygenase that incorporates both oxygens of O₂ into formic acid. (A) Reaction of FlcD in a ¹⁶O₂ or ¹⁸O₂ environment. (B, C) LC-HRMS spectra of the FlcD product, 3, from a reaction using ¹⁶O-2 in an ¹⁸O₂ environment or using ¹⁸O-2 in a ¹⁶O₂ environment. (D) Reaction of [¹³C₂,¹⁵N]-2 in a ¹⁶O₂ or ¹⁸O₂ environment. (E) GC-HRMS spectra of formic acid produced in the FlcD reaction in a ¹⁶O₂ (red) or ¹⁸O₂ (blue) environment. Reaction in an ¹⁸O₂ environment produced double ¹⁸O-labeled formic acid. (F) Reaction of FlcD using [2,3,3-D₃]-2 as substrate. (G) LC-HRMS spectrum of the FlcD product, 3, showing that one deuterium is retained in 3 during the reaction (F). H) GC-HRMS spectrum shows enrichment of a deuterium in formic acid using [2,3,3-D₃,¹³C₃,¹⁵N]-2 as a substrate for FlcD. LC-HRMS analysis was performed using electrospray ionization under positive ion mode. GC-HRMS analysis was performed using electron ionization under positive ion mode. All observed m/z from GC-HRMS analysis is within 1 ppm error of the calculated m/z.

Figure 5

Proposed mechanism for oxidative cleavage catalyzed by FlcD. Fe is coordinated by E181, H191, and H274 and three waters. Relevant substrate atoms are labeled: carbon 2 of cysteine (yellow dot), the oxime oxygen (red), O₂ (blue), and deuterium (green). FlcD begins with a mononuclear Fe(II) cofactor (A). Substrate replaces one of the water molecules and coordinates to Fe in a monodentate fashion (B). O₂ is activated as Fe(III)-superoxo (C), which abstracts a deuterium from carbon 3, generating a radical (D). Hydroxylation at carbon 2 results in formation of an Fe(IV)-oxo, and radical recombination leads to formation of a hydroxy aziridine ring (E). Ring opening of the hydroxy aziridine results in an aldehyde (F). Two-electron oxidation of the hydroxylamine by Fe(IV)-oxo generates the nitroso and Fe(II) (G). Fe(II)-hydroxyl is transferred to the aldehyde. Isomerization of the nitroso and elimination of carbon 2 lead to formation of the oxime product, 3, and formic acid (H). Products are released, completing the catalytic cycle.