Directed Evolution of a Cp*Rh^III‐Linked Biohybrid Catalyst Based on a Screening Platform with Affinity Purification

Abstract

Directed evolution of Cp*RhIII‐linked nitrobindin (NB), a biohybrid catalyst, was performed based on an in vitro screening approach. A key aspect of this effort was the establishment of a high‐throughput screening (HTS) platform that involves an affinity purification step employing a starch‐agarose resin for a maltose binding protein (MBP) tag. The HTS platform enables efficient preparation of the purified MBP‐tagged biohybrid catalysts in a 96‐well format and eliminates background influence of the host E. coli cells. Three rounds of directed evolution and screening of more than 4000 clones yielded a Cp*RhIII‐linked NB(T98H/L100K/K127E) variant with a 4.9‐fold enhanced activity for the cycloaddition of acetophenone oximes with alkynes. It is confirmed that this HTS platform for directed evolution provides an efficient strategy for generating highly active biohybrid catalysts incorporating a synthetic metal cofactor.

Chemogenetic Evolution of a Peroxidase-like Artificial Metalloenzyme

“…An iron-depleted minimal medium was used to avoid unwanted incorporation of FePPIX during the expression. , After cell lysis, 3 equiv of the MnPPIX cofactor were supplemented to form the ArM (typical scaffold protein concentrations were ∼20 μM in cleared lysates; Figure S3). Previously, ArMs were successfully immobilized on chromatography resins to streamline screening procedures. − Therefore, the mixture was applied onto a Strep-Tactin resin and washed twice using a 96-well filter plate. Finally, the Strep-Tactin immobilized ArM variants were transferred into a transparent MTP and their peroxidase activity was determined using ABTS as the substrate and AcOOH as the oxidant.…”

Designing Artificial Metalloenzymes by Tuning of the Environment beyond the Primary Coordination Sphere

“…In addition to Fe and Cu metalloenzymes, there are many ArMs that have employed other biologically relevant metal cofactors, such as V, Cr, Mn, ,, Co, , Ni, , Zn, and Mg, as well as those with abiological metal centers, including Ru, Rh, ,,, Pd, Os, Ir, ,,, and Au . These studies can be divided into two categories, namely those that target understanding native metalloenzyme functionality and those that aim to utilize the ArM scaffold to enhance the catalytic properties of organometallic complexes.…”

“…Directed evolution was later applied to this system, and the T98H/L100K/K127E mutant was identified to increase the activity 2.2-fold. Interestingly, MD simulations suggested that all three of these residues are located opposite to the Rh metal center …”

“…Interestingly, MD simulations suggested that all three of these residues are located opposite to the Rh metal center. 594 Human carbonic anhydrase II (hCA II) exhibits a high binding affinity toward para-substituted aryl-sulfonamides at the Zn binding site, providing another tool for the development of ArMs. An {IrCp*} 2+ moiety was introduced into hCA II to generate a novel ATHase.…”

Evolutionary Engineering of a Cp*Rh(III) Complex-Linked Artificial Metalloenzyme with a Chimeric β-Barrel Protein Scaffold

“…34−36 A series of engineered variants of NB-1 generated via SSM were found to catalyze cycloaddition of acetophenone oxime (2a) and 1,4-dimethoxy-2-butyne (3a) via C(sp 2 )−H bond activation (Figure 1c). 36 However, these point mutations had only minor effects on the catalytic activity because the Cp*Rh(III) cofactor of NB-1 was largely exposed to solvent due to its shallow cavity. It was also speculated that the existence of an H 2 O molecule in the active-site limits the catalytic performance of the artificial metalloenzymes in general.…”