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 Ltyrosine 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.

Nitric oxide synthase-guided genome mining identifies a cytochrome P450 enzyme for olefin nitration in bacterial specialized metabolism

“…Subsequently, a cytochrome P450 enzyme (TxtE) was shown to catalyze tryptophan nitration using NO and O 2 [ 10 ]. Similar aromatic nitration mediated by a NOS/P450 pair (RufN/RufO or IlaM/IlaN) was also found in the biosynthesis of rufomycins/ilamycins [ [11] , [12] , [13] , [14] ]. In addition, we recently showed that the N8 atom in the unique 1,2,3-triazole heterocycle of the antiviral agent 8-azaguanine derives from NO, which is synthesized by a NOS (PtnF) [ 15 ].…”

Nitration of a ribosomal pentapeptide generates a noncanonical precursor for nonribosomal peptide synthesis

“…Our investigations into the function of RufO stemmed from comparisons between its structure (23,24) and that of the biarylitide crosslinking P450Blt that we had recently characterized (26). Analysis of these structures revealed that they were highly similar (41.6% amino acid ID, C-a RMSD 1.8 Å to 8SPC; Figure S6), apart from mutations at a key section of the RufO I-helix involved in oxygen activation (27).…”

“…Due to the similarity of the genes rufNO to the txtDE system, which has been demonstrated to perform nitration of tryptophan in thaxtomin biosynthesis (13), the rufomycin 3-NO2-Tyr residue has been postulated as being synthesized by an analogous mechanism involving direct nitration of free Tyr (15). However, this hypothesis is not supported by reported data, and recent studies suggest RufO instead acts on Tyr bound to a carrier protein domain following selection and activation by the NRPS machinery (22)(23)(24).…”