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

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.