Crystal Structure and Conformational Dynamics of N─N Bond-Forming Piperazate Synthase

l-Piperazic acid (l-Piz) is a noncanonical, α-hydrazino acid characterized by a 1,2-diazinane heterocycle containing an N─N bond. It occurs in numerous natural products with potent biological activities and represents a key pharmaceutical building block. In nature, l-Piz is biosynthesized from l-ornithine via the intermediate N⁵-hydroxy-l-ornithine in a two-enzyme cascade comprising a flavin adenine dinucleotide (FAD)-dependent N-hydroxylating monooxygenase (NMO) and a heme-dependent piperazate synthase (PZS). The NMO selectively hydroxylates the δ-amino group of l-ornithine, while PZS catalyzes intramolecular N─N bond formation to generate the six-membered cyclic hydrazine scaffold of l-Piz. Here, we report the crystal structure, Piz-forming activity, and molecular dynamics (MD) analysis of SbPZS, a representative PZS from Streptomyces sp. B93. High-resolution structural analysis enabled a detailed comparison with previously characterized PZS homologs. To further delineate the molecular basis of catalysis, we performed MD simulations in combination with sequence-based bioinformatic analyses. These studies provide insight into protein-substrate interactions, conformational dynamics, and the residues that contribute to active-site organization. Moreover, we identify candidate hotspots for engineering to modulate substrate scope and catalytic efficiency. Collectively, our results establish a structural framework for understanding enzymatic N─N bond formation in Piz biosynthesis and lay the groundwork for future biocatalytic applications of PZSs.