Structural and spectroscopic characterization of RufO indicates a new biological role in rufomycin biosynthesis

Abstract

Rufomycins constitute a class of cyclic heptapeptides isolated from actinomycetes. They are secondary metabolites that show promising treatment against Mycobacterium tuberculosis infections by inhibiting a novel drug target. Several nonproteinogenic amino acids are integrated into rufomycins, including a conserved 3-nitro-tyrosine. RufO, a cytochrome P450 (CYP)-like enzyme, was proposed to catalyze the formation of 3-nitro-tyrosine in the presence of O₂ and NO. To define its biological function, the interaction between RufO and the proposed substrate tyrosine is investigated using various spectroscopic methods that are sensitive to the structural change of a heme center. However, a low- to high-spin state transition and a dramatic increase in the redox potential that are commonly found in CYPs upon ligand binding have not been observed. Furthermore, a 1.89-Å crystal structure of RufO shows that the enzyme has flexible surface regions, a wide-open substrate access tunnel, and the heme center is largely exposed to solvent. Comparison with a closely related nitrating CYP reveals a spacious and hydrophobic distal pocket in RufO, which is incapable of stabilizing a free amino acid. Molecular docking validates the experimental data and proposes a possible substrate. Collectively, our results disfavor tyrosine as the substrate of RufO and point to the possibility that the nitration occurs during or after the assembly of the peptides. This study indicates a new function of the unique nitrating enzyme and provides insights into the biosynthesis of nonribosomal peptides.

Keywords: cytochrome P450; heme-dependent catalysis; nonribosomal peptide synthesis; rufomycin; tyrosyl nitration.

Figure 1

RufO is invovled in the biosynthesis of rufomycins.A, general structure of rufomycins. 3-NO₂-Tyr is highlighted. Positions R₁-R₄ are widely modified in rufomycins. B, nitration reaction catalyzed by RufO. R is an amino acid moiety proposed by a previous study (11).

Figure 2

Measurements of reduction potential and activity of RufO.A, Nernst plot of RufO enzyme alone (black) and enzyme with 1 mM Tyr (red). The data points are derived from the absorption spectra upon reduction shown in Fig. S2. Linear fittings yielded y-intercepts (ΔE_m = E_RufO -E_dye) of −1 and −2, respectively. B, HPLC profile of RufO activity assay. The traces from top to bottom are HPLC analysis of 1 mM Tyr standard, 1 mM 3-NO₂-Tyr standard, the reaction under condition 1 using chemically reduced enzyme, and the reaction under condition 2 using PdR/PdX redox pair. Peaks eluted around 3, 4, and 13 min are excess NADH, Tyr, and 3-NO₂-Tyr, respectively. PdR, putidareductase; PdX, putidaredoxin.

Figure 3

Spectroscopic analysis of RufO heme center in the presence and absence of 2 mM Tyr.A, absorption spectra of oxidized RufO enzyme alone (black) and enzyme with 2 mM Tyr (red). Inset: spectra of reduced RufO enzyme alone (gray) and enzyme with 2 mM Tyr (blue). B, X-band continuous wave EPR spectra of oxidized RufO enzyme alone (black) and enzyme with 2 mM Tyr (red). The spectra were collected at 30 K with a microwave power of 1.0 mW. C, resonance Raman spectra of RufO enzyme alone (black) and enzyme with 2 mM Tyr (red). The spectra were collected using the 413.1 nm laser line at 5 mW. EPR, electron paramagnetic resonance.

Figure 4

A 1.89-Å crystal structure of RufO. (A) top view and (B) side view of the overall structure and (C) the heme proximal site. The cartoon representation is colored by a rainbow spectrum from a blue N-terminus to a red C-terminus. The α-helices, β-sheets, and 3₁₀ helices are noted. The N, O, S, Fe, and heme C atoms are in blue, red, yellow, brown, and deep red, respectively. The 2F_o–F_c difference maps of the heme center are colored in light blue and contoured at 1.0 σ. The gray dashed lines indicate distances (Å) between atoms. Regions 1 and 2 showing the largest structural discrepancies when compared with TxtE and CYPs are marked by black circles. CYP, cytochrome P450.

Figure 5

Comparison of RufO and TxtE structures.A, vertical cross sections (left) and top views (right) of RufO (top) and TxtE (bottom) in surface representation. Regions 1 and 2 are colored in blue and yellow, respectively. Water molecules in the substrate access channels are shown in red spheres in the top views. The PDB entry of the TxtE structure is 4L36. B, distal pockets of RufO (top) and TxtE (bottom). Color code of atoms: protein carbon, white; tryptophan carbon, yellow; heme carbon, deep red; iron, brown; nitrogen, blue; oxygen, red. Gray dashed lines indicate distances (Å) between atoms. Active site water molecules are removed for clarity. The PDB entry of the tryptophan-bound TxtE structure is 4TPO. PDB, Protein Data Bank.

Figure 6

Molecular docking of RufO. The docking poses and 2D ligand–protein interaction diagrams for RufO docked with (A and B) tyrosine and (C and D) a cyclic peptide. Tyr, the cyclic peptide, heme, and RufO are colored in blue, orange, red, and white, respectively. In the 2D diagrams, purple arrows represent the hydrogen bonding; green curves represent the π–π stacking; gray shaded atoms are solvent exposed; colored droplets represent heme (gray), negatively charged (red), positively charged (blue), polar uncharged (cyan), and hydrophobic residues (green).