Isonitrile Formation by a Non-Heme Iron(II)-Dependent Oxidase/Decarboxylase

Abstract

The electron-rich isonitrile is an important functionality in bioactive natural products, but its biosynthesis has been restricted to the IsnA family of isonitrile synthases. We herein provide the first structural and biochemical evidence of an alternative mechanism for isonitrile formation. ScoE, a putative non-heme iron(II)-dependent enzyme from Streptomyces coeruleorubidus, was shown to catalyze the conversion of (R)-3-((carboxymethyl)amino)butanoic acid to (R)-3-isocyanobutanoic acid through an oxidative decarboxylation mechanism. This work further provides a revised scheme for the biosynthesis of a unique class of isonitrile lipopeptides, of which several members are critical for the virulence of pathogenic mycobacteria.

Keywords: acyl-acyl carrier protein ligase; biosynthesis; isocyanide; oxidoreductase; protein structures.

Figure 1.

Schematic of isonitrile lipopeptide biosynthetic pathway. This revised pathway shows that ScoE utilizes a free acid substrate, 4, and after isonitrile formation, the product 5 is reactivated and loaded onto ScoB by ScoC.

Figure 2.

Structure of ScoE (PDB 6DCH) compared to TauD (PDB 1OS7). A) Overall structure of a ScoE protomer shown in yellow ribbon representation. The metal-coordinating 2-His-1-Asp facial triad and a conserved active site Arg are shown in yellow ball-and-stick representation. Acetate and choline ligands within the active site are shown in green ball-and-stick. Only one of the two observed conformations of choline is shown for clarity. Zn(II) and Cl⁻ are shown as gray and green spheres, respectively. B) Overall structure of a TauD protomer shown in teal ribbon representation and oriented similarly to ScoE in A). The metal-coordinating 2-His-1-Asp facial triad and a conserved active site Arg are shown in teal ball-and-stick representation. Taurine and αKG within the active site are shown in tan ball-and-stick. Fe(II) is shown as an orange sphere. C) Zoomed in view of the active site of ScoE. Composite omit density is shown in Figure S7. D) Active site of TauD displayed similarly to C).

Figure 3.

In vitro characterization of ScoE. A) Extracted ion chromatograms showing the conversion of 4 to 5 catalyzed by ScoE. For simplicity, only the no αKG assay is shown as a representative negative control. The calculated mass of 5 with a 10-ppm mass error tolerance was used. B) Extracted ion chromatograms showing the production of 2. Bottom trace displays the assay with 1 and ScoE. Top trace shows the coupled reaction containing ScoE, ScoB, ScoC and 4. The calculated mass of 2 with a 10-ppm mass error tolerance was used. The deconvoluted mass spectrum of 2 is displayed on the right. C) Extracted ion chromatograms showing the formation of 3 in the total enzymatic synthesis using ScoA, ScoB, ScoC, ScoE and 4. The calculated mass of 3 with a 10-ppm mass error tolerance was used.