A novel catalytic heme cofactor in SfmD with a single thioether bond and a bis-His ligand set revealed by a de novo crystal structural and spectroscopic study

Abstract

SfmD is a heme-dependent enzyme in the biosynthetic pathway of saframycin A. Here, we present a 1.78 Å resolution de novo crystal structure of SfmD, which unveils a novel heme cofactor attached to the protein with an unusual Hx _n HxxxC motif (n ∼ 38). This heme cofactor is unique in two respects. It contains a single thioether bond in a cysteine-vinyl link with Cys317, and the ferric heme has two axial protein ligands, i.e., His274 and His313. We demonstrated that SfmD heme is catalytically active and can utilize dioxygen and ascorbate for a single-oxygen insertion into 3-methyl-l-tyrosine. Catalytic assays using ascorbate derivatives revealed the functional groups of ascorbate essential to its function as a cosubstrate. Abolishing the thioether linkage through mutation of Cys317 resulted in catalytically inactive SfmD variants. EPR and optical data revealed that the heme center undergoes a substantial conformational change with one axial histidine ligand dissociating from the iron ion in response to substrate 3-methyl-l-tyrosine binding or chemical reduction by a reducing agent, such as the cosubstrate ascorbate. The labile axial ligand was identified as His274 through redox-linked structural determinations. Together, identifying an unusual heme cofactor with a previously unknown heme-binding motif for a monooxygenase activity and the structural similarity of SfmD to the members of the heme-based tryptophan dioxygenase superfamily will broaden understanding of heme chemistry.

Fig. 1. SfmD is an ascorbate- and O₂-dependent monooxygenase. (A) The catalytic reaction of SfmD, (B) HPLC analysis of the hydroxylation of 3-Me-l-Tyr catalyzed by SfmD. Absorbance at 280 nm was monitored and presented with the same scale. Peaks in the red and blue shaded boxes correspond to substrate and product, respectively. The reaction conditions were (i) 3-Me-l-Tyr, (ii) SfmD and 3-Me-l-Tyr, (iii) SfmD, 3-Me-l-Tyr, and H₂O₂, (iv) SfmD, 3-Me-l-Tyr, H₂O₂, and ascorbate, (v) SfmD, 3-Me-l-Tyr, and ascorbate under anaerobic condition, (vi) SfmD, 3-Me-l-Tyr, and ascorbate under air-saturated conditions, (vii) SfmD, 3-Me-l-Tyr, and ascorbate under O₂-saturation. The concentrations were: 30 μM enzyme, 1 mM substrate 3-Me-l-Tyr, 1 mM H₂O₂, and 20 mM ascorbate, (C) the maximum absorbance (λ_max) of the substrate 3-Me-l-Tyr (red color trace) and reaction product 3-OH-5-Me-l-Tyr (blue), and (D) positive-mode ESI mass spectra for the enzyme reaction product. Mass spectra are shown in range 211 ≤ m/z ≤ 216. Experimental mass of 3-OH-5-Me-l-Tyr matches the calculated one with 0.47 ppm mass accuracy.

Fig. 2. Ascorbate slowly reduces SfmD. (A) UV-vis spectra of SfmD (black trace) and SfmD with ascorbate (blue trace), (B) the same as (A) except with the presence of 3-Me-l-Tyr, (C) UV-vis spectra of SfmD with ascorbate (black trace) and SfmD with ascorbate and ˙NO (blue trace), and (D) the same as (C) except with the presence of 3-Me-l-Tyr. The plots for changes at 425 nm as a function of time are shown in the inset in panels (A)–(D).

Fig. 3. Investigation of the structure-based specificity for ascorbate as a cosubstrate during catalysis. (A) Ascorbate derivatives were employed in this study, (B) HPLC analysis of the hydroxylation of l-Tyr catalyzed by SfmD. Absorbance at 280 nm was monitored and presented with the same scale. Peaks in the red and blue shaded boxes correspond to substrate and product, respectively. The reaction conditions were (i) l-Tyr, (ii) SfmD, l-Tyr, and ascorbate, (iii) SfmD, l-Tyr, and 3-O-ethyl-l-ascorbate, (iv) SfmD, l-Tyr, and 2-O-α-d-glucopyranosyl-l-ascorbate, (v) SfmD, l-Tyr, and 5,6-isopropylidene-l-ascorbate, (vi) SfmD, l-Tyr, and l-dehydroascorbate, (vii) SfmD, l-Tyr, and l-ascorbate 2-phosphate, (C) the same as (B) except executed reaction with ferrous SfmD. The concentrations were: 30 μM enzyme, 1 mM l-Tyr, and 20 mM ascorbate and its derivatives.

Fig. 4. The three-dimensional structure of SfmD showing a novel heme cofactor and the heme-binding motif. (A) The overall structure of SfmD (PDB entry: 6VDQ) is shown in two different orientations. Helices in the N-terminal and C-terminal half are presented in blue and white, respectively. Heme is shown in the red space-filling model, (B) A single cysteine–vinyl thioether bond, and a bis-His axial metal–ligand motif revealed a novel heme prosthetic group. Cys317 covalently connects to the heme, whereas Cys234 does not. (C) 2F_o − F_c electron density maps from the final structural refinement are presented in blue contoured at 1σ. Omit F_o − F_c electron density maps for heme and Cys317 after simulated annealing are shown in orange contoured at 3σ. (D) Amino acid sequence alignment of SfmD and the hypothetical SfmD-like proteins is shown with the secondary structural elements of SfmD. Asterisks represent the residues involved in heme binding: His274, His313, and Cys317 in SfmD. (E) Superposition of the core structures of SfmD with TDO (PDB entry: 2NOX). SfmD is shown in white and cyan color for helices and loops with a heme (pink) bound. TDO is presented in blue and orange color for helices and salmon color for loops with a heme (red). Orange helices are the secondary structural elements not present in SfmD. Parentheses denote the secondary structure elements of TDO.

Fig. 5. A distal heme ligand of SfmD swings off from the iron ion in response to substrate binding or chemical reduction. (A) EPR spectra of SfmD (200 μM, black trace) and SfmD after addition of 3-Me-l-Tyr (2.5 mM, red trace), (B) EPR spectra of ferric SfmD (gray trace), after addition of ascorbate and ˙NO (black), and after addition of 3-Me-l-Tyr, ascorbate, and ˙NO (blue). The EPR spectra of the ferric SfmD were measured at 10 K, while the nitrosyl complexes were measured at 50 K (see ESI, Materials and methods).

Fig. 6. Partially reduced crystal structure of SfmD identifies iron ligand His274 rotating out during active site reorganization. (A) Single-crystal UV-vis spectroscopy of SfmD (black trace) after in crystallo reduction by dithionite in the presence of 5 mM 3-Me-l-Tyr (blue and green traces for partially reduced crystals, and red trace for the reduced crystal that did not diffract), and (B) a partially reduced structure determined from the crystal that yielded the green spectrum in (A) and aligned against the SfmD structure in the resting state (with carbon atoms and main chains in gray color). The carbon atoms and the main chain of the partially reduced structure are shown in green with heme in dark red. Electron density maps for the conformational change in the partially reduced structures are shown for (C) 6VDZ and (D) 6VE0. 2F_o − F_c electron density maps from the final structural refinement are presented in blue contoured at 1σ. Omit F_o − F_c electron density maps for the heme ligand His274 and the loop region containing (267–271, green) after simulated annealing are shown in orange contoured at 3σ. Electron density maps are shown in two different orientations. A movie clip showing more detailed structural rearrangements is available in the ESI.

Fig. 7. Mutation of Cys317 abolishes the covalency attachment of the heme and catalytic activity. (A) UV-vis spectra of SfmD C317S (black trace) and C317S SfmD with ascorbate (blue trace); (B) the same as (A) except with the presence of 3-Me-l-Tyr; (C) UV-vis spectra of C317S with ascorbate (black trace) and C317S with ascorbate and ˙NO (blue trace); (D) the same as (C) except with the presence of 3-Me-l-Tyr. The plots for changes at 425 nm as a function of time are shown in the inset in panels (A)–(D); (E) EPR spectra of C317S (black trace), C317S after addition of 3-Me-l-Tyr (red trace), and C317S after addition of ascorbate (blue trace); and (F) EPR spectra of ferric C317S (gray trace), after addition of ascorbate and ˙NO (black), or after addition of 3-Me-l-Tyr, ascorbate, and ˙NO (blue).