Indexado en

Licencia y uso

Citaciones

Altmetrics

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).

Análisis de autorías institucional

Compartir

Directed Evolution of an Artificial Hydroxylase Based on a Thermostable Human Carbonic Anhydrase Protein

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

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

Afiliaciones

Resumen

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.

Palabras clave