Insights into Substrate Recognition by the Unusual Nitrating Enzyme RufO

Abstract

Nitration reactions are crucial for many industrial syntheses; however, current protocols lack site specificity and employ hazardous chemicals. The noncanonical cytochrome P450 enzymes RufO and TxtE catalyze the only known direct aromatic nitration reactions in nature, making them attractive model systems for the development of analogous biocatalytic and/or biomimetic reactions that proceed under mild conditions. While the associated mechanism has been well-characterized in TxtE, much less is known about RufO. Herein we present the first structure of RufO alongside a series of computational and biochemical studies investigating its unusual reactivity. We demonstrate that free l-tyrosine is not readily accepted as a substrate despite previous reports to the contrary. Instead, we propose that RufO natively modifies l-tyrosine tethered to the peptidyl carrier protein of a nonribosomal peptide synthetase encoded by the same biosynthetic gene cluster and present both docking and molecular dynamics simulations consistent with this hypothesis. Our results expand the scope of direct enzymatic nitration reactions and provide the first evidence for such a modification of a peptide synthetase-bound substrate. Both of these insights may aid in the downstream development of biocatalytic approaches to synthesize rufomycin analogues and related drug candidates.

Figure 1

Structural comparison of RufO (PDB entry 8SPC) and l-Trp-bound TxtE (PDB entry 4TPO). (A) Overall fold of RufO with annotated α-helices and β-sheets. Note that β6 is too small to depict. (B) Loop regions facilitating access to the active site. (C) RufO and (D) TxtE active site residues surrounding the catalytic heme cofactor. RufO and TxtE α-helices/β-sheets/loops are shown in blue/gold/gray and red/green/tan, respectively. The heme cofactor is colored white and l-Trp in light green.

Figure 2

(A) UV–vis absorption spectra of substrate-free RufO in the ferric (black) and ferrous (gray) states. Q bands are magnified in the inset. (B) Spectral components representing the ferric superoxo (red) and NO-bound (blue) states of RufO determined via singular value decomposition and global fitting of stopped-flow data.

Figure 3

Absorption spectra and activity assays demonstrating the lack of significant l-Tyr binding to RufO. (A) Soret peaks and Q bands are unaltered by l-Tyr binding (black) and only shift following the addition of 1 M HCl (purple). (B) Direct-injection mass spectra of RufO reactions with l-Tyr (red peak). The red diamond indicates where 3-nitro-l-Tyr is expected (m/z [M + H]⁺ = 227.032). (C) Absorption spectra of RufO bound to l-Tyr analogues selected to mimic the amino acid bound to the phosphopantetheinyl arm of the PCP domain. Spectral traces have been offset for clarity. (D, E) Corresponding mass spectra from direct injection assessing reactivity with (D) TME or (E) TEEM. Red peaks correspond to these small molecules, while red diamonds indicate where nitrated products are expected (TME-NO₂: m/z [M + H]⁺ = 242.082; TEEM-NO₂: m/z [M + H]⁺ = 298.109).

Figure 4

Computational prediction of the RufO substrate complex. (A) Surface model of the apo PCP domain bound to RufO. We highlight the serine that is ultimately phosphopantetheinylated in red. (B) Time-evolution RMSD plots of three independent 50 ns MD simulations of RufO in complex with the docked phosphopantetheinyl arm covalently linked to l-Tyr. (C) Snapshots of the three trajectories bound in the RufO active site pocket. Variation is primarily observed where the arm would attach to the PCP domain. (D) Representative configuration of the PCP-bound l-Tyr primed for the nitration reaction. The distance between the Fe ion and C3 is highlighted, as are key hydrogen bonds and hydrophobic contacts. See Figure S14 for a more complete interaction diagram. Secondary structure elements are colored as in Figure 1.