Structure and mechanism of the azomycin biosynthetic enzyme RohQ that catalyzes a spontaneous cyclodehydration

RohQ from the azomycin biosynthetic pathway catalyzes a spontaneous cyclodehydration to form 2-aminoimidazole. Here we report the structure and mechanism of RohQ and use a serendipitously bound imidazole to pinpoint active site residues. We propose that catalysis occurs at the dimeric interface using two key aspartic acid residues for proton transfer steps to accelerate 3 $\times$ 104-fold intramolecular cyclization of a guanidino group and aldehyde, releasing water. Our work expands our understanding of de novo emerged enzymes and provides the first structural and mechanistic view of a yet-unexplored protein family.