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PreprintCC BYChemRxiv

Comparative Characterization of Nitrogen–Nitrogen Bond-Forming Piperazate Synthases to Improve Activity and Precision

  • Simon Schröder
  • Lan Julij Zadravec
  • Nikita Pal1,2
  • Angelina Osipyan
  • Hanna Mozer
  • Simon P. Godehard
  • Alexander Pelzer
  • Zvjezdana Findrik Blažević
  • Karl Gruber
  • Martin A. Hayes
  • Sandy Schmidt
  1. 1Karl Gruber Institute of Molecular Biosciences
  2. 2University of Graz Humboldtstraße
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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.

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Cite

BibTeXRIS
BibTeX
@misc{schroder2026comparative,
  title = {{Comparative Characterization of Nitrogen–Nitrogen Bond-Forming Piperazate Synthases to Improve Activity and Precision}},
  author = {Simon Schröder and Lan Julij Zadravec and Nikita Pal and Angelina Osipyan and Hanna Mozer and Simon P. Godehard and Alexander Pelzer and Zvjezdana Findrik Blažević and Karl Gruber and Martin A. Hayes and Sandy Schmidt},
  howpublished = {ChemRxiv},
  year = {2026},
  month = sep,
  doi = {10.26434/chemrxiv.15009069/v1},
  url = {https://chemrxiv.org/doi/full/10.26434/chemrxiv.15009069/v1},
  note = {CC BY},
}
RIS
TY  - UNPB
TI  - Comparative Characterization of Nitrogen–Nitrogen Bond-Forming Piperazate Synthases to Improve Activity and Precision
AU  - Simon Schröder
AU  - Lan Julij Zadravec
AU  - Nikita Pal
AU  - Angelina Osipyan
AU  - Hanna Mozer
AU  - Simon P. Godehard
AU  - Alexander Pelzer
AU  - Zvjezdana Findrik Blažević
AU  - Karl Gruber
AU  - Martin A. Hayes
AU  - Sandy Schmidt
PY  - 2026
DA  - 2026/09/18
PB  - ChemRxiv
DO  - 10.26434/chemrxiv.15009069/v1
UR  - https://chemrxiv.org/doi/full/10.26434/chemrxiv.15009069/v1
AB  - 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.
ER  -