A Pseudokinase Catalyzes Nitrile Formation in the Biosynthesis of a Potent Marine Toxin

ABSTRACT Several pseudokinases, previously regarded as dead enzymes due to the lack of catalytic residues, catalyze nucleotidylation. While they often utilize macromolecular substrates such as proteins and RNAs in primary metabolism, those acting on non‐macromolecules in specialized metabolisms are limited. Calyculin A, a cytotoxic natural product produced by an uncultured sponge symbiont, possesses a unique nitrile group at the end of its tetraene tail. Even though its biosynthetic gene cluster (BGC) has been identified, the enzyme responsible for nitrile formation remains unknown. Herein, through a comparative analysis of the BGCs for calyculin derivatives in symbiotic bacteria from distinct sources, we identified a novel nitrile‐forming enzyme, CalN. While CalN lacks sequence homology with other known nitrile‐forming enzymes, it is structurally similar to pseudokinases. In vitro enzymatic reactions demonstrated that CalN specifically catalyzes nitrile formation through the adenylation of an amide substrate, calyculinamide A. In silico analyses and mutational experiments showed that CalN's structure features a unique insertion that plays critical roles in ATP recognition and the spatial coordination of catalytic residues. This study not only identifies a new family of nitrile‐forming enzymes but also expands the variety of chemical reactions mediated by pseudokinases in nature.