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Grant support

This publication was created as part of NCCR Catalysis (grant no. 180544), a National Centre of Competence in Research funded by the Swiss National Science Foundation. Additional funding was provided by the NCCR Molecular Systems Engineering (grant 200021_178760) and the SNSF (grant 200020_212088). The authors thank the Swiss Light Source and Diamond Light Source for X-ray data collection and X. Li-Blatter for her help in data fitting for the binding assays and differential scanning fluorimetry experiments. I.M. thanks the Nakajima Foundation for a PhD stipend. A.F. thanks Ministerio de Universidades for a short-term mobility funding from the predoctoral fellowship FPU19/05872 (grant ref EST21/00132).

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Faraone, AdrianaAuthor

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November 19, 2024
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Directed Evolution of an Artificial Hydroxylase Based on a Thermostable Human Carbonic Anhydrase Protein

Publicated to:Acs Catalysis. - 2024-11-07 (), DOI: 10.1021/acscatal.4c04163

Authors: Morita, Iori; Faraone, Adriana; Salvisberg, Elias; Zhang, Kailin; Jakob, Roman P; Maier, Timm; Ward, Thomas R

Affiliations

Barcelona Inst Sci & Technol, ICIQ Inst Chem Res Catalonia, Tarragona 43007, Spain - Author
Ecole Polytech Fed Lausanne, Inst Sci & Ingn Chim, CH-1015 Lausanne, Switzerland - Author
IST Austria, Campus 1, A-3400 Klosterneuburg, Austria - Author
Univ Basel, Biozentrum, CH-4056 Basel, Switzerland - Author
Univ Basel, Dept Chem, CH-4002 Basel, Switzerland - Author
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Abstract

The assembly of artificial metalloenzymes (ArMs) provides a second coordination sphere around a metal catalyst. Such a well-defined microenvironment can lead to an enhancement of the activities and selectivity of the catalyst. Herein, we present the development of artificial hydroxylase (ArHase) by embedding an Fe-TAML (TAML = tetraamide macrocyclic ligand) catalyst into a human carbonic anhydrase II (hCAII). Incorporation of the Fe-TAML catalyst ([BS-Fe-bTAML]-) within hCAII enhanced the total turnover number (TTON) for the hydroxylation of benzylic C-H bonds. After engineering a thermostable variant of hCAII (hCAIITS), the resulting ArHase, [BS-Fe-bTAML]-hCAIITS, was subjected to directed evolution using cell lysates in a 384-well format. After three rounds of laboratory evolution, the best-performing variants exhibited enhancement in the initial rate (124.4 min-1) and in the TTON (2629 TTON) for the hydroxylation of benzylic C-H bonds compared to that of the free cofactor. We surmise that an arginine residue introduced in the course of directed evolution engages in hydrogen bonding with [BS-Fe-bTAML]-. This study highlights the potential of relying on a thermostable host protein to improve the catalytic performance of hCAII-based ArMs.

Keywords

Artificial metalloenzymeAssaBindingCatalysisComplexCoordinationDesignDirected evolutionEarth-abundant metalEnzymeFunctionalizationHydroxylationLigandMetalloenzymesNonheme ironcatalys

Quality index

Bibliometric impact. Analysis of the contribution and dissemination channel

The work has been published in the journal Acs Catalysis due to its progression and the good impact it has achieved in recent years, according to the agency WoS (JCR), it has become a reference in its field. In the year of publication of the work, 2024 there are still no calculated indicators, but in 2023, it was in position 21/178, thus managing to position itself as a Q1 (Primer Cuartil), in the category Chemistry, Physical.

Impact and social visibility

From the perspective of influence or social adoption, and based on metrics associated with mentions and interactions provided by agencies specializing in calculating the so-called "Alternative or Social Metrics," we can highlight as of 2025-07-06:

  • The use, from an academic perspective evidenced by the Altmetric agency indicator referring to aggregations made by the personal bibliographic manager Mendeley, gives us a total of: 8.
  • The use of this contribution in bookmarks, code forks, additions to favorite lists for recurrent reading, as well as general views, indicates that someone is using the publication as a basis for their current work. This may be a notable indicator of future more formal and academic citations. This claim is supported by the result of the "Capture" indicator, which yields a total of: 4 (PlumX).

With a more dissemination-oriented intent and targeting more general audiences, we can observe other more global scores such as:

  • The Total Score from Altmetric: 0.5.
  • The number of mentions on the social network X (formerly Twitter): 1 (Altmetric).

Leadership analysis of institutional authors

This work has been carried out with international collaboration, specifically with researchers from: Austria; Switzerland.