Probing the Mechanism of Isonitrile Formation by a Non-Heme Iron(II)-Dependent Oxidase/Decarboxylase

Abstract

The isonitrile moiety is an electron-rich functionality that decorates various bioactive natural products isolated from diverse kingdoms of life. Isonitrile biosynthesis was restricted for over a decade to isonitrile synthases, a family of enzymes catalyzing a condensation reaction between l-Trp/l-Tyr and ribulose-5-phosphate. The discovery of ScoE, a non-heme iron(II) and α-ketoglutarate-dependent dioxygenase, demonstrated an alternative pathway employed by nature for isonitrile installation. Biochemical, crystallographic, and computational investigations of ScoE have previously been reported, yet the isonitrile formation mechanism remains obscure. In the present work, we employed in vitro biochemistry, chemical synthesis, spectroscopy techniques, and computational simulations that enabled us to propose a plausible molecular mechanism for isonitrile formation. Our findings demonstrate that the ScoE reaction initiates with C5 hydroxylation of (R)-3-((carboxymethyl)amino)butanoic acid to generate 1, which undergoes dehydration, presumably mediated by Tyr96 to synthesize 2 in a trans configuration. (R)-3-isocyanobutanoic acid is finally generated through radical-based decarboxylation of 2, instead of the common hydroxylation pathway employed by this enzyme superfamily.

Figure 1.

Overall reaction of ScoE and detection of possible intermediates. (A) Extracted ion chromatograms (EICs) showing glyoxylate and R-3-ABA formation from ScoE assays derivatized with 2-NPH under acidic conditions. Glyoxylate and R-3-ABA are degradation products of possible reaction intermediate 2 under acidic conditions. (B) EICs of 3 generated from the ScoE reaction subject to negative controls. (C) EICs demonstrating production of 4 from assays containing CABA, α-KG, Fe(II), and ScoE. Metabolite 4 shows no evidence of consumption from the time course. A 10-ppm mass error tolerance was used for each trace with the masses listed.

Figure 2.

Biochemical analysis of ScoE utilizing various substrates. (A) EICs demonstrating the production of 3,5-di(pyridine-2-yl)-1Hpyrazol-4-amine (Py-AP) as a product of the isonitriletetrazine click reaction when 2 was used as a substrate. Omission of any of the assay components led to abolition of Py-AP. Utilization of 3 and 5 as substrates was not recognized by ScoE. (B) ScoE kinetic parameters for 2 in INBA formation. The data points and error bars represent the average and standard deviation from three independent experiments, respectively. Calculated mass for Py-AP (m/z=238.1088 [M+H]⁺) was used for each trace with a 10-ppm error tolerance.

Figure 3.

LC-MS quantification and kinetic rate constants for ScoE reaction products. A) Relative amounts of INBA, 2, 3, and 4 quantified by LC-MS from a series of in vitro assays using different ratios of CABA:α-KG (1:1 and 1:3). Error bars correspond to standard deviation of the mean from three replicate experiments. Detailed procedures are found in the Supporting Information. B) Apparent rate constants of formation at excess α-KG conditions (1:3) for compounds 2, 3, and 4. Error bars correspond to standard deviation of the mean from three replicate experiments.

Figure 4.

Depiction of the ScoE active site and the amino acids selected for inclusion in the QM cluster model. Key residues, ligands, and interactions are shown with their skeleton structures. Interactions are shown as dotted red lines. All residues and ligands are superimposed on the ScoE backbone with the α-carbon positions denoted with a black dot.

Figure 5.

Relative energy profiles in kcal/mol for the first and second half reactions with optimized intermediate structures. (A) The first half reaction depicts the energy levels of the first C5-H HAT and the subsequent hydroxyl rebound, as well as the final generation of 2. (B) The second half reaction depicts the energy levels of the second HAT from 2 and the decarboxylation triggered by a radical transfer to the iron center. The intermediates 1 and 2 are labeled in bold. Only the coordinating atoms and substrate are shown for clarity.

Scheme 1.

Proposed mechanism for isonitrile formation by ScoE.