Structural and Mechanistic Basis for Nitrile Synthetase by an Argininosuccinate Synthetase-Like Enzyme

The enzymatic origins of nitrile groups in fungal natural products have recently been linked to argininosuccinate synthetase (ASS)-like enzymes, but the structural basis for their unique catalytic function remained unknown. Here, we provide the structural and mechanistic elucidation of this enzyme class by characterizing ArtA, the nitrile synthetase from the auranthine biosynthetic pathway. High-resolution crystal structures of ArtA, combined with mutagenesis and molecular dynamics simulations, reveal how the canonical ASS active site is remodeled to selectively bind l-glutamine and catalyze nitrile formation through an elegant ATP-dependent elimination reaction. Furthermore, phylogenetic analysis and functional validation of homologues from another fungus (Fusarium) and a bacterium (Streptomyces) demonstrate that ArtA belongs to a widespread family of nitrile synthetases, likely acquired by fungi via horizontal gene transfer. Our work provides the molecular blueprint for this new enzyme family and opens avenues for genome mining and biocatalyst engineering.