Liu, X.; Wang, J.; Wu, Y.; Zhong, F.

J. Am. Chem. Soc. 2021, 143, 617-622, 10.1021/jacs.0c10882

Devising artificial photoenzymes for abiological bond-forming reactions is of high synthetic value but also a tremendous challenge. Disclosed herein is the first photobiocatalytic cross-coupling of aryl halides enabled by a designer artificial dehalogenase, which features a genetically encoded benzophenone chromophore and site-specifically modified synthetic NiII(bpy) cofactor with tunable proximity to streamline the dual catalysis. Transient absorption studies suggest the likelihood of energy transfer activation in the elementary organometallic event. This design strategy is viable to significantly expand the catalytic repertoire of artificial photoenzymes for useful organic transformations.

DeGrado, W.F.

J. Am. Chem. Soc. 2021, 143, 252-259, 10.1021/jacs.0c10136

De novo protein design offers the opportunity to test our understanding of how metalloproteins perform difficult transformations. Attaining high-resolution structural information is critical to understanding how such designs function. There have been many successes in the design of porphyrin-binding proteins; however, crystallographic characterization has been elusive, limiting what can be learned from such studies as well as the extension to new functions. Moreover, formation of highly oxidizing high-valent intermediates poses design challenges that have not been previously implemented: (1) purposeful design of substrate/oxidant access to the binding site and (2) limiting deleterious oxidation of the protein scaffold. Here we report the first crystallographically characterized porphyrin-binding protein that was programmed to not only bind a synthetic Mn–porphyrin but also maintain binding site access to form high-valent oxidation states. We explicitly designed a binding site with accessibility to dioxygen units in the open coordination site of the Mn center. In solution, the protein is capable of accessing a high-valent Mn(V)–oxo species which can transfer an O atom to a thioether substrate. The crystallographic structure is within 0.6 Å of the design and indeed contained an aquo ligand with a second water molecule stabilized by hydrogen bonding to a Gln side chain in the active site, offering a structural explanation for the observed reactivity.

Pordea, A.

J. Inorg. Biochem. 2021, 220, 111446, 10.1016/j.jinorgbio.2021.111446

Artificial metalloenzymes result from the insertion of a catalytically active metal complex into a biological scaffold, generally a protein devoid of other catalytic functionalities. As such, their design requires efforts to engineer substrate binding, in addition to accommodating the artificial catalyst. Here we constructed and characterised artificial metalloenzymes using alcohol dehydrogenase as starting point, an enzyme which has both a cofactor and a substrate binding pocket. A docking approach was used to determine suitable positions for catalyst anchoring to single cysteine mutants, leading to an artificial metalloenzyme capable to reduce both natural cofactors and the hydrophobic 1-benzylnicotinamide mimic. Kinetic studies revealed that the new construct displayed a Michaelis-Menten behaviour with the native nicotinamide cofactors, which were suggested by docking to bind at a surface exposed site, different compared to their native binding position. On the other hand, the kinetic and docking data suggested that a typical enzyme behaviour was not observed with the hydrophobic 1-benzylnicotinamide mimic, with which binding events were plausible both inside and outside the protein. This work demonstrates an extended substrate scope of the artificial metalloenzymes and provides information about the binding sites of the nicotinamide substrates, which can be exploited to further engineer artificial metalloenzymes for cofactor regeneration.

Hayashi, T; Onoda, A.

ChemBioChem 2021, 22, 679-685, 10.1002/cbic.202000681

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.

Lewis, J.C.; Roux, B.

Angew. Chem. Int. Ed. 2021, 60, 23672-23677, 10.1002/anie.202107982

Artificial metalloenzymes (ArMs) are commonly used to control the stereoselectivity of catalytic reactions, but controlling chemoselectivity remains challenging. In this study, we engineer a dirhodium ArM to catalyze diazo cross-coupling to form an alkene that, in a one-pot cascade reaction, is reduced to an alkane with high enantioselectivity (typically >99 % ee) by an alkene reductase. The numerous protein and small molecule components required for the cascade reaction had minimal effect on ArM catalysis. Directed evolution of the ArM led to improved yields and E/Z selectivities for a variety of substrates, which translated to cascade reaction yields. MD simulations of ArM variants were used to understand the structural role of the cofactor on ArM conformational dynamics. These results highlight the ability of ArMs to control both catalyst stereoselectivity and chemoselectivity to enable reactions in complex media that would otherwise lead to undesired side reactions.

Jarvis, A.G.

ACS Catal. 2021, 11, 3620-3627, 10.1021/acscatal.0c05413

Protein engineering has shown widespread use in improving the industrial application of enzymes and broadening the conditions they are able to operate under by increasing their thermostability and solvent tolerance. Here, we show that protein engineering can be used to increase the thermostability of an artificial metalloenzyme. Thermostable variants of the human steroid carrier protein 2L, modified to bind a metal catalyst, were created by rational design using structural data and a 3DM database. These variants were tested to identify mutations that enhanced the stability of the protein scaffold, and a significant increase in melting temperature was observed with a number of modified metalloenzymes. The ability to withstand higher reaction temperatures resulted in an increased activity in the hydroformylation of 1-octene, with more than fivefold improvement in turnover number, whereas the selectivity for linear aldehyde remained high up to 80%.

Lombardi, A.; Louro, R.O.

Chem. Commun. 2021, 57, 990-993, 10.1039/d0cc06676a

Fe-Mimochrome VI*a is a synthetic peroxidase and peroxygenase, featuring two different peptides that are covalently-linked to deuteroheme. To perform a systematic structure/function correlation, we purposely shortened the distance between the distal peptide and the heme, allowing for the separation and characterization of two regioisomers. They differ in both His axial-ligand orientation, as determined by paramagnetic NMR shifts, and activity. These findings highlight that synthetic metalloenzymes may provide an efficient tool for disentangling the role of axial ligand orientation over peroxidase activity.

Anderson, J.L.R.

J. Inorg. Biochem. 2021, 217, 111370, 10.1016/j.jinorgbio.2021.111370

The design and construction of de novo enzymes offer potentially facile routes to exploiting powerful chemistries in robust, expressible and customisable protein frameworks, while providing insight into natural enzyme function. To this end, we have recently demonstrated extensive catalytic promiscuity in a heme-containing de novo protein, C45. The diverse transformations that C45 catalyses include substrate oxidation, dehalogenation and carbon‑carbon bond formation. Here we explore the substrate promiscuity of C45's peroxidase activity, screening the de novo enzyme against a panel of peroxidase and dehaloperoxidase substrates. Consistent with the function of natural peroxidases, C45 exhibits a broad spectrum of substrate activities with selectivity dictated primarily by the redox potential of the substrate, and by extension, the active oxidising species in peroxidase chemistry, compounds I and II. Though the comparison of these redox potentials provides a threshold for determining activity for a given substrate, substrate:protein interactions are also likely to play a significant role in determining electron transfer rates from substrate to heme, affecting the kinetic parameters of the enzyme. We also used biomolecular simulation to screen substrates against a computational model of C45 to identify potential interactions and binding sites. Several sites of interest in close proximity to the heme cofactor were discovered, providing insight into the catalytic workings of C45.