{
  "abstract": "Enzymatic N–N bond formation is a rare and chemically challenging transformation in natural product biosynthesis. The non-heme iron metalloenzyme SznF catalyzes the formation of the N -nitrosourea pharmacophore of streptozotocin via the coordinated action of a diiron heme-oxygenase-like (HDO) domain and a mononuclear iron cupin domain. Although recent computational studies have advanced mechanistic models for this transformation, the determinants governing substrate specificity within this enzyme family remain poorly understood. Here, we employed a multi-pronged approach combining genome mining, substrate scope screening, protein engineering, structural analysis, and biochemical characterization to define the catalytic boundaries of SznF and related homologs. Genome mining identified a previously uncharacterized homolog displaying higher activity toward the native substrate N ω -methyl-L-arginine than SznF, expanding the known repertoire of N-nitrosourea-forming metalloenzymes. Comprehensive substrate screening further revealed the first non-native substrate accepted by this enzyme family, N ω -propyl-L-arginine, whereas all other modifications in carbon chain length, guanidino substitution, alkylation state, or stereochemistry did not result in detectable turnover. Structure-guided engineering that targeted the binding pocket surrounding the terminal alkyl substituent increased the activity of several variants toward N ω -propyl-L-arginine compared to wildtype SznF. Furthermore, cascade experiments, and nitric oxide supplementation further demonstrate that productive catalysis requires a precise combination of steric, electronic, and geometric features that cannot readily be compensated by alternative substrates. Together, these findings show that the substrate specificity of SznF and related homologs is governed by mechanistic constraints arising from the tightly coupled hydroxylation and rearrangement chemistry underlying N–N bond formation. More broadly, this work defines the catalytic boundaries of enzymatic N -nitrosourea formation while identifying new opportunities for the discovery and engineering of N–N bond-forming metalloenzymes for biocatalytic synthesis of structurally diverse N -nitrosourea derivatives.",
  "abstract_shown": true,
  "affiliations": [
    "Groningen Research Institute of Pharmacy, University of Groningen, Antonius Deusinglaan 1, Groningen, Netherlands, 9713AV"
  ],
  "article_type": null,
  "arxiv": null,
  "author_total": 4,
  "authors": [
    {
      "affs": [
        0
      ],
      "name": "Charitomeni Angeli"
    },
    {
      "affs": [
        0
      ],
      "name": "Mohammad Faizan Bhat"
    },
    {
      "affs": [
        0
      ],
      "name": "Frank J. Dekker"
    },
    {
      "affs": [
        0
      ],
      "name": "Sandy Schmidt",
      "orcid": "0000-0002-8443-8805"
    }
  ],
  "bibtex": "https://research.draconitic.com/paper/4gmk43w1ynne1.bib",
  "cited_by": 0,
  "doi": "10.26434/chemrxiv.15010285/v1",
  "fulltext_url": null,
  "id": "4gmk43w1ynne1",
  "issns": [],
  "landing_url": "https://chemrxiv.org/doi/full/10.26434/chemrxiv.15010285/v1",
  "language": null,
  "licence": "CC-BY",
  "licence_label": "CC BY",
  "licence_source": "journal",
  "licence_url": "https://creativecommons.org/licenses/by/4.0/",
  "metadata_licence": null,
  "native_subjects": [
    [
      "openalex",
      "2736|Microbial Natural Products and Biosynthesis"
    ]
  ],
  "openalex": "W7221569898",
  "pdf_url": null,
  "pmcid": null,
  "pmid": null,
  "published": "2026-10-12",
  "publisher": "American Chemical Society (ACS)",
  "ris": "https://research.draconitic.com/paper/4gmk43w1ynne1.ris",
  "sources": [
    {
      "first_seen": "2026-10-30T12:17:22Z",
      "native_id": "10.26434/chemrxiv.15010285",
      "source": "chemrxiv",
      "url": "https://chemrxiv.org/doi/full/10.26434/chemrxiv.15010285/v1"
    }
  ],
  "subjects": [
    {
      "field": "medicine",
      "origin": "openalex:2736|Microbial Natural Products and Biosynthesis",
      "subfield": "pharmacology-toxicology"
    }
  ],
  "title": "Defining the Catalytic Boundaries of N -Nitrosourea-Forming StzF/SznF and Related Metalloenzymes through Genome Mining and Mechanistic Analysis",
  "title_text": "Defining the Catalytic Boundaries of N -Nitrosourea-Forming StzF/SznF and Related Metalloenzymes through Genome Mining and Mechanistic Analysis",
  "type": "preprint",
  "updated_at": "2026-10-30T12:17:22Z",
  "url": "https://research.draconitic.com/paper/4gmk43w1ynne1",
  "venue": "ChemRxiv",
  "versions": []
}