Structural Basis for the Catalytic Mechanism of ATP‐Dependent Diazotase CmaA6

Abstract Although several diazotases have been recently reported, the details of the reaction mechanism are not yet understood. In this study, we investigated the mechanism of CmaA6, an ATP‐dependent diazotase, which catalyzes the diazotization of 3‐aminocoumaric acid using nitrous acid. X‐ray crystallography and cryogenic electron microscopy‐single particle analysis revealed CmaA6 structures in the substrate‐free and AMP‐binding states. Kinetic analysis suggested that CmaA6 catalyzes diazotization via a sequential reaction mechanism in which three substrates (nitrous acid, ATP, and 3‐aminocoumaric acid) are simultaneously bound in the reaction pocket. The nitrous acid and 3‐aminocoumaric acid binding sites were predicted based on the AMP‐binding state and confirmed by site‐directed mutagenesis. In addition, computational analysis revealed a tunnel for 3‐aminocoumaric acid to enter the reaction pocket, which was advantageous for the sequential reaction mechanism. This study provides important insights into the catalytic mechanism of diazotization in natural product biosynthesis.