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 alpha-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 N-N bonds, have been identified thus far. The hemoenzyme KtzT from Kutzneria sp. 744 catalyzes the intramolecular N-N 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 N-N bond formation indicated a considerable thermo- and pH-flexibility, with optimal reaction conditions at 30 degrees 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 hemeand 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.