Enhancing biocatalytical N-N bond formation with the actinobacterial piperazate synthase KtzT

Natural compounds with nitrogen-nitrogen bonds are diverse and have applications in medicine and agriculture. l-Piperazic acid (Piz), an $-hydrazino acid, is one of few naturally occurring compounds of its kind. Yet, Piz and its derivatives are valuable building blocks for bioactive compounds. Few NNzymes, enzymes capable of forming NN bonds, have been identified thus far. The hemoenzyme KtzT from Kutzneria sp. 744 catalyzes the intramolecular NN bond formation of N5‑hydroxy-l-ornithine (OH-Orn) to form Piz, a natural building block of kutznerides. The latter has antifungal and antibiotic properties. In our study, we established an improved expression method, with significantly improved yields (ca. 35-fold) of heme-loaded enzyme, making the enzyme much more accessible for laboratory studies. In vitro biochemical characterization under conditions for NN bond formation indicated a considerable thermo- and pH-flexibility, with optimal reaction conditions at 30 °C and 10 mM Tris buffer at pH 9 together with low salinity, paving the way for more complex applications involving KtzT. We have also identified two homologous enzymes from extremophilic organisms to exhibit piperazate-forming activity. In silico structural studies, combined with phylogenetic analysis, resulted in a heme- and substrate-binding model, suggesting target enzyme residues that we propose are critical for the structural integrity and catalytic activity of KtzT. Following this approach, we investigated the potential role of a cysteine residue in a dimer-stabilizing disulfide bridge. The interplay of in vitro and in silico data therefore provides crucial functional information on this enzyme class.