{
  "abstract": "Natural enzyme families achieve functional breadth by diversifying a common scaffold into specialized enzymes. Engineering comparable family-like diversity for synthetic chemistry is challenging because directed evolution workflows frequently optimize one enzyme for one substrate at a time. Here we show that an iterative process of designing and screening energetically compatible multimutant variants can explore sequence space broadly and efficiently. Starting from weak azole N-methylation by human nicotinamide N-methyltransferase, screening only 1,600 clones over three rounds yielded an enzyme ensemble with broad scope. The generated catalysts carried up to 11 active-site substitutions and displayed mean initial-rate gains equivalent to approximately 11-fold per round, with overall gains exceeding 3,000-fold for individual enzyme-substrate pairs. Regioselectivity exceeded 99:1, preparative methylation afforded 87% isolated yield, and all thermally characterized variants were more thermostable than the wild type. Broad activity across diverse azoles emerged collectively from sequence-diverse specialist enzymes. Mapping all mutational routes between two high-activity variants showed that each passed through a lessactive intermediate, revealing why stepwise local optimization can miss high-performing variants. Thus, iteratively testing energetically compatible multimutant designs transformed one promiscuous enzyme into a family-like ensemble of complementary catalysts.",
  "abstract_shown": true,
  "affiliations": [
    "Faculty of Chemistry",
    "Bielefeld University"
  ],
  "article_type": null,
  "arxiv": null,
  "author_total": 9,
  "authors": [
    {
      "affs": [
        0,
        1
      ],
      "name": "Lucas Bocquin"
    },
    {
      "affs": [
        0,
        1
      ],
      "name": "Om Desai"
    },
    {
      "affs": [
        0,
        1
      ],
      "name": "Kateryna Zelenska"
    },
    {
      "affs": [
        0,
        1
      ],
      "name": "Matthias Garbrecht"
    },
    {
      "affs": [
        0,
        1
      ],
      "name": "Tom Hoenig"
    },
    {
      "affs": [
        0,
        1
      ],
      "name": "Alina Klein"
    },
    {
      "affs": [
        0,
        1
      ],
      "name": "Friederike Reese"
    },
    {
      "affs": [
        0,
        1
      ],
      "name": "Kai Schülke"
    },
    {
      "name": "Stephan Christian Hammer",
      "orcid": "0000-0002-3620-9362"
    }
  ],
  "bibtex": "https://research.draconitic.com/paper/1aj5ybsawdg13.bib",
  "cited_by": 0,
  "doi": "10.26434/chemrxiv.15009747/v1",
  "fulltext_url": null,
  "id": "1aj5ybsawdg13",
  "issns": [],
  "landing_url": "https://chemrxiv.org/doi/full/10.26434/chemrxiv.15009747/v1",
  "language": null,
  "licence": "CC-BY-NC-ND",
  "licence_label": "CC BY-NC-ND",
  "licence_source": "journal",
  "licence_url": "https://creativecommons.org/licenses/by-nc-nd/4.0/",
  "metadata_licence": null,
  "native_subjects": [
    [
      "openalex",
      "1312|Enzyme Catalysis and Immobilization"
    ]
  ],
  "openalex": "W7215039102",
  "pdf_url": null,
  "pmcid": null,
  "pmid": null,
  "published": "2026-10-01",
  "publisher": "American Chemical Society (ACS)",
  "ris": "https://research.draconitic.com/paper/1aj5ybsawdg13.ris",
  "sources": [
    {
      "first_seen": "2026-10-30T12:17:21Z",
      "native_id": "10.26434/chemrxiv.15009747",
      "source": "chemrxiv",
      "url": "https://chemrxiv.org/doi/full/10.26434/chemrxiv.15009747/v1"
    }
  ],
  "subjects": [
    {
      "field": "biochemistry",
      "origin": "openalex:1312|Enzyme Catalysis and Immobilization",
      "subfield": "molecular-biology"
    }
  ],
  "title": "Iterative Multimutant Design Creates a Family-Like Enzyme Ensemble",
  "title_text": "Iterative Multimutant Design Creates a Family-Like Enzyme Ensemble",
  "type": "preprint",
  "updated_at": "2026-10-30T12:17:21Z",
  "url": "https://research.draconitic.com/paper/1aj5ybsawdg13",
  "venue": "ChemRxiv",
  "versions": []
}