Pathway from N‐Alkylglycine to Alkylisonitrile Catalyzed by Iron(II) and 2‐Oxoglutarate‐Dependent Oxygenases

Abstract

N-alkylisonitrile, a precursor to isonitrile-containing lipopeptides, is biosynthesized by decarboxylation-assisted -NC group (isonitrile) formation by using N-alkylglycine as the substrate. This reaction is catalyzed by iron(II) and 2oxoglutarate (Fe/2OG) dependent enzymes. Distinct from typical oxygenation or halogenation reactions catalyzed by this class of enzymes, installation of the isonitrile group represents a novel reaction type for Fe/2OG enzymes that involves a four-electron oxidative process. Reported here is a plausible mechanism of three Fe/2OG enzymes, Sav607, ScoE and SfaA, which catalyze isonitrile formation. The X-ray structures of iron-loaded ScoE in complex with its substrate and the intermediate, along with biochemical and biophysical data reveal that -NC bond formation involves two cycles of Fe/2OG enzyme catalysis. The reaction starts with an Fe IV -oxocatalyzed hydroxylation. It is likely followed by decarboxylation-assisted desaturation to complete isonitrile installation.

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

“…We obtained several crystal structures of ScoE with and without substrates and noted an ordered water network extending from the CABA site. , Through site-directed mutagenesis, we confirmed the importance of Tyr96, Tyr97, Tyr101, and Arg195 at maintaining the hydrogen-bonding network between three ordered water molecules because alterations of these residues completely abrogated or severely compromised isonitrile formation. Several pathways to isonitrile formation were previously proposed; however, a definitive pathway with evidence for reaction intermediates remains elusive. ,, …”

“…We next sought to assign 1–5 to be reaction intermediates or shunt products. 1 was proposed to be the first intermediate in the pathway based on the previously reported structure of ScoE with 1 and detection of 1 from a 13 C NMR experiment analyzing an enzymatic assay from SfaA . Our recent structures of ScoE with bound CABA further demonstrated one of the C5 hydrogen points toward the iron cofactor, suggesting that C5 was the most likely site of initial C–H activation and hydroxylation .…”

“… , An in vitro biochemical assay incubated for 3 h at 4 °C containing 5- 13 C-CABA, resulted in the detection of two new enriched correlations in HSQC and one new enriched correlation in HMBC, respectively, which were not present in assays containing unlabeled CABA (Figures S4 and S5). The first HSQC signal at δc ∼ 88 ppm was consistent with the C5 signal of C5–OH CABA ( 1 ) that was previously reported for an enzymatic assay with SfaA, a homologue of ScoE (85% sequence similarity) . The second HSQC signal detected at δc∼162 ppm was assigned to C5 of imine CABA ( 2 ) in comparison to a synthetic standard (Figures S5A and S7).…”

“…Similarly, a biochemical assay using synthetic 5 did not generate isonitrile, suggesting that 5 was also not an intermediate (Figure A). The failed recognition of 5 by ScoE is in agreement with a previous work by Chen et al, but Chen favored 3 as an on-pathway intermediate, which is in contrast to our observations . We could not synthesize a standard of 4 for ScoE assays due to its inherent propensity to tautomerize to 5 or become hydrolyzed.…”

Molecular insights into the unusually promiscuous and catalytically versatile Fe(II)/α-ketoglutarate-dependent oxygenase SptF

“…Substrate scope investigation of SptF. a-c, LC-MS EICs for in vitro assays using preandiloid B (10), preandiloid C (12), terretonin J (13), terretonin C (15), terretonin A (17),and terretonin(19). The + m/z value used for each compound is shown.…”