Iterative Multimutant Design Creates a Family-Like Enzyme Ensemble
- 1Faculty of Chemistry
- 2Bielefeld University
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.
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BibTeX
@misc{bocquin2026iterative,
title = {{Iterative Multimutant Design Creates a Family-Like Enzyme Ensemble}},
author = {Lucas Bocquin and Om Desai and Kateryna Zelenska and Matthias Garbrecht and Tom Hoenig and Alina Klein and Friederike Reese and Kai Schülke and Stephan Christian Hammer},
howpublished = {ChemRxiv},
year = {2026},
month = oct,
doi = {10.26434/chemrxiv.15009747/v1},
url = {https://chemrxiv.org/doi/full/10.26434/chemrxiv.15009747/v1},
note = {CC BY-NC-ND},
}
RIS
TY - UNPB
TI - Iterative Multimutant Design Creates a Family-Like Enzyme Ensemble
AU - Lucas Bocquin
AU - Om Desai
AU - Kateryna Zelenska
AU - Matthias Garbrecht
AU - Tom Hoenig
AU - Alina Klein
AU - Friederike Reese
AU - Kai Schülke
AU - Stephan Christian Hammer
PY - 2026
DA - 2026/10/01
PB - ChemRxiv
DO - 10.26434/chemrxiv.15009747/v1
UR - https://chemrxiv.org/doi/full/10.26434/chemrxiv.15009747/v1
AB - 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.
ER -