Argininosuccinate Synthetase-Like Enzyme Drives Nitrile Formation in Fungal Natural Products Biosynthesis

Nitrile groups are vital components of diverse bioactive molecules, however, their enzymatic origins in fungal natural products remain largely unknown. Here, we report the discovery of an argininosuccinate synthetase (AsS)-like enzyme, ArtA, which plays a key role in the biosynthesis of a nitrile-containing nonribosomal peptide, auranthine. Structural and mechanistic studies, including X-ray crystallography, site-directed mutagenesis, docking, and molecular dynamics simulations, reveal that ArtA retains core AsS features, but possesses a remodeled active site that accommodates glutamine and facilitates nitrile formation through a unique mechanism. Furthermore, we demonstrated that ArtA homologs are prevalent across fungi and bacteria, and also demonstrated nitrile synthetase activity of representative homologs from both fungi and bacteria, suggesting a broader distribution of this function. The identification of ArtA and its homologs opens new avenues for genome mining and biotechnological applications targeting nitrile-containing natural products.