Shaw, W.J.

Organometallics 2020, 39, 1532-1544, 10.1021/acs.organomet.9b00843

The protein scaffold around the active site of enzymes is known to influence catalytic activity, but specific scaffold features responsible for favorable influences are often not known. This study focuses on using an artificial metalloenzyme to probe one specific feature of the scaffold, the position of a positive charge in the outer coordination sphere around the active site. Previous work showed that a small molecular complex, [Rh(PEt2NglycinePEt2)2]−, immobilized covalently within a protein scaffold was activated for CO2 hydrogenation. Here, using an iterative design where the effect of arginine, histidine, or lysine residues placed in the outer coordination sphere of the catalytic active site were evaluated, we tested the hypothesis that positively charged groups facilitate CO2 hydrogenation with seven unique constructs. Single-, double-, and triple-point mutations were introduced to directly compare catalytic activity, as monitored by turnover frequencies (TOFs) measured in real time with 1H NMR spectroscopy, and evaluate related structural and electronic properties. Two of the seven constructs showed a 2- and 3-fold increase relative to the wild type, with overall rates ranging from 0.2 to 0.7 h–1, and a crystal structure of the fastest of these shows the positive charge positioned next to the active site. A crystal structure of the arginine-containing complex shows that the arginines are positioned near the metal. Molecular dynamics (MD) studies also suggest that the positive charge is oriented next to the active site in the two constructs with faster rates but not in the others and that the positive charge near the active site holds the CO2 near the metal, all consistent with a positive charge appropriately positioned in the scaffold benefiting catalysis. The MD studies also suggest that changes in the water distribution around the active site may contribute to catalytic activity, while modest structural changes and movement of the complex within the scaffold do not.

Jeschek, M.

Trends Biotechnol. 2018, 36, 60-72, 10.1016/j.tibtech.2017.10.003

Residing at the interface of chemistry and biotechnology, artificial metalloenzymes (ArMs) offer an attractive technology to combine the versatile reaction repertoire of transition metal catalysts with the exquisite catalytic features of enzymes. While earlier efforts in this field predominantly comprised studies in well-defined test-tube environments, a trend towards exploiting ArMs in more complex environments has recently emerged. Integration of these artificial biocatalysts in enzymatic cascades and using them in whole-cell biotransformations and in vivo opens up entirely novel prospects for both preparative chemistry and synthetic biology. We highlight selected recent developments with a particular focus on challenges and opportunities in the in vivo application of ArMs.

Seelig, B.

Trends Biotechnol. 2010, 28, 340-345, 10.1016/j.tibtech.2010.04.003

Enzymes offer cheap, environmentally responsible and highly efficient alternatives to chemical catalysts. The past two decades have seen a significant rise in the use of enzymes in industrial settings. Although many natural enzymes have been modified through protein engineering to better suit practical applications, these approaches are often insufficient. A key goal of enzyme engineers is to build enzymes de novo – or, ‘from scratch’. To date, several technologies have been developed to achieve this goal: namely, computational design, catalytic antibodies and mRNA display. These methods rely on different principles, trading off rational protein design against an entirely combinatorial approach of directed evolution of vast protein libraries. The aim of this article is to review and compare these methods and their potential for generating truly de novo biocatalysts.

Eppinger, J.

ChemistryOpen 2013, 2, 50-54, 10.1002/open.201200044

How to train a protein: Metal‐conjugated affinity labels were used to selectively position catalytically active metal centers in the binding pocket of proteases. The resulting artificial metalloenzymes achieve up to 82 % e.r. in the hydrogenation of ketones. The modular setup enables a rapid generation of artificial metalloenzyme libraries, which can be adapted to a broad range of catalytic conditions.