Comparative Characterization of Nitrogen–Nitrogen Bond-Forming Piperazate Synthases to Improve Activity and Precision
Year: 2026
Type: preprint
Abstract: Piperazate synthases (PZSs) have recently gained attention as enzymes that catalyze the formation of nitrogen-nitrogen bonds, offering a sustainable alternative to traditional synthesis of substituted hydrazines. Although progress has been made in elucidating their structure and mechanism, an exhaustive study characterizing multiple PZSs regarding their activity or tendency toward side reactions under varying conditions has yet to be conducted. In this study, we profiled the activity of eight purified PZSs under 80 different conditions each, covering pH, buffers, salinity, temperature dependence, and thermal stability, using automated liquid handling and a colorimetric substrate consumption assay. We also performed kinetic and structural analysis of the two most promising PZSs and used LC-MS to quantitatively investigate the substrate scope and side reactions. Among the investigated enzymes, we identified two highly active synthases, SbPZS from Streptomyces sp. B93 and SgPZS from Streptomyces griseochromogenes. Both enzymes tolerated a broad range of reaction conditions, but differed in their side-product formation rates. We found this tendency to be sequence-dependent and controlled by pH. These findings provide detailed information regarding the in vitro applicability of PZSs and will improve their use in (chemo-)enzymatic cascades for synthesizing pharmaceutical building blocks.
Source: ChemRxiv
Institution: University of Graz
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: green
Awards: G3951938444, G5956220919
OpenAlex ID: W7213557131