Cited by 4Protein Structure and DynamicsEnzyme Structure and FunctionEnzyme Catalysis and Immobilization

A De Novo Designed Protein with Versatile Metal Binding and Tunable Hydrolytic Activity

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Hoffnagle, Alexander M., Srisantitham, Suppachai, Neeley, Maximilian, Tsai, Chia-Ying, Tezcan, F. Akif·2025·Biochemistry

Published version of a preprint

Abstract

Metalloenzyme superfamilies achieve diverse functions within a shared structural framework, and similar functional variety may be achievable in designed proteins. We have previously reported a computational approach that enables the de novo design of symmetric protein assemblies around metal centers with pre-defined coordination geometries. Here, we demonstrate that an artificial protein trimer termed Tet4, whose structure was designed around an idealized tetrahedral His3/H2O-ZnII coordination motif, enables the high-affinity binding of several other divalent first-row transition metal ions in the same geometry as for ZnII. We then follow the proposed evolutionary path of a natural metalloenzyme family by engineering a pseudosymmetric, single chain variant of Tet4, scTet425. scTet425 allows us to introduce asymmetric point mutations that influence the catalytic properties of the metal center. We also demonstrate that we can further tune the enzymatic activity of Tet4 by designing a substrate pocket that improves Zn-Tet4’s affinity for a hydrolysis substrate, 4-methylumbelliferyl acetate.

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Crossref · 10.1021/acs.biochem.5c00259

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Authors
Hoffnagle, A. M., Srisantitham, S., Neeley, M., Tsai, C.-Y., & Tezcan, F. A.
Affiliations
University of California San Diego (US)
Corresponding author
Tezcan, F. Akif
Venue
Biochemistry
Published
2025-07-23
Volume
64
Issue
15
Pages
3261-3271
ISSN
  • 0006-2960 (ISSN-L)
  • 1520-4995
OpenAlex
W4412610193
Cited by
4
References
82
Citations by year
2026: 3 · 2025: 1
FWCI
1.20
Type
journal article
Language
en
Open access
Locations
  • Biochemistry (publishedVersion)
  • ChemRxiv (acceptedVersion, cc-by)
  • PubMed (publishedVersion)
Funders
National Institute of General Medical Sciences (12948080, R01-GM138884)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein Structure and Dynamics
  • Physical Sciences › Materials Science › Materials Chemistry › Enzyme Structure and Function
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Enzyme Catalysis and Immobilization
Keywords
de novo protein design, metalloenzymes, zinc binding, tetrahedral coordination, protein trimer
MeSH terms
Binding Sites, Hydrolysis, Metalloproteins, Metals, Models, Molecular, Protein Binding, Zinc, Protein Engineering

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