Gao, X.; Zhao, L.

Sci. Adv. 2020, 6, 10.1126/sciadv.abb1421

Metalloenzymes are promising anticancer candidates to overcome chemoresistance by involving unique mechanisms. To date, it is still a great challenge to obtain synthetic metalloenzymes with persistent catalytic performance for cancer-specific DNA cleavage and operando imaging. Here, an artificial metalloenzyme, copper cluster firmly anchored in bovine serum albumin conjugated with tumor-targeting peptide, is exquisitely constructed. It is capable of persistently transforming hydrogen peroxide in tumor microenvironment to hydroxyl radical and oxygen in a catalytic manner. The stable catalysis recycling stems from the electron transfer between copper cluster and substrate with well-matched energy levels. Notably, their high biocompatibility, tumor-specific recognition, and persistent catalytic performance ensure the substantial anticancer efficacy by triggering DNA damage. Meanwhile, by coupling with enzyme-like reactions, the operando therapy effect is expediently traced by chemiluminescence signal with high sensitivity and sustainability. It provides new insights into synthesizing biocompatible metalloenzymes on demand to visually monitor and efficiently combat specific cancers.

Anderson, J.L.R.

Nat. Commun. 2017, 8, 10.1038/s41467-017-00541-4

Although catalytic mechanisms in natural enzymes are well understood, achieving the diverse palette of reaction chemistries in re-engineered native proteins has proved challenging. Wholesale modification of natural enzymes is potentially compromised by their intrinsic complexity, which often obscures the underlying principles governing biocatalytic efficiency. The maquette approach can circumvent this complexity by combining a robust de novo designed chassis with a design process that avoids atomistic mimicry of natural proteins. Here, we apply this method to the construction of a highly efficient, promiscuous, and thermostable artificial enzyme that catalyzes a diverse array of substrate oxidations coupled to the reduction of H2O2. The maquette exhibits kinetics that match and even surpass those of certain natural peroxidases, retains its activity at elevated temperature and in the presence of organic solvents, and provides a simple platform for interrogating catalytic intermediates common to natural heme-containing enzymes.

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.

Anderson, J.L.R.

Curr. Opin. Struct. Biol. 2018, 51, 149-155, 10.1016/j.sbi.2018.04.008

Though established 40 years ago, the field of de novo protein design has recently come of age, with new designs exhibiting an unprecedented level of sophistication in structure and function. With respect to catalysis, de novo enzymes promise to revolutionise the industrial production of useful chemicals and materials, while providing new biomolecules as plug-and-play components in the metabolic pathways of living cells. To this end, there are now de novo metalloenzymes that are assembled in vivo, including the recently reported C45 maquette, which can catalyse a variety of substrate oxidations with efficiencies rivalling those of closely related natural enzymes. Here we explore the successful design of this de novo enzyme, which was designed to minimise the undesirable complexity of natural proteins using a minimalistic bottom-up approach.

Soumillion, P.

ChemBioChem 2006, 7, 1013-1016, 10.1002/cbic.200600127

Enantioselective epoxidation of styrene was observed in the presence of manganese‐containing carbonic anhydrase as catalyst. The probable oxygen‐transfer reagent is peroxymonocarbonate, which has a structural similarity with the hydrogenocarbonate substrate of the natural reaction. Styrene was chosen as the enzyme possesses a small hydrophobic cavity close to the active site.

Clark, D.S.; Hartwig, J.F.; Keasling, J.D.; Mukhopadhyay, A.

Nat. Chem. 2021, 13, 1186-1191, 10.1038/s41557-021-00801-3

Synthetic biology enables microbial hosts to produce complex molecules from organisms that are rare or difficult to cultivate, but the structures of these molecules are limited to those formed by reactions of natural enzymes. The integration of artificial metalloenzymes (ArMs) that catalyse unnatural reactions into metabolic networks could broaden the cache of molecules produced biosynthetically. Here we report an engineered microbial cell expressing a heterologous biosynthetic pathway, containing both natural enzymes and ArMs, that produces an unnatural product with high diastereoselectivity. We engineered Escherichia coli with a heterologous terpene biosynthetic pathway and an ArM containing an iridium–porphyrin complex that was transported into the cell with a heterologous transport system. We improved the diastereoselectivity and product titre of the unnatural product by evolving the ArM and selecting the appropriate gene induction and cultivation conditions. This work shows that synthetic biology and synthetic chemistry can produce, by combining natural and artificial enzymes in whole cells, molecules that were previously inaccessible to nature.