A Cell-Penetrating Artificial Metalloenzyme Regulates a Gene Switch in a Designer Mammalian Cell
Nat. Commun. 2018, 9, 10.1038/s41467-018-04440-0
Complementing enzymes in their native environment with either homogeneous or heterogeneous catalysts is challenging due to the sea of functionalities present within a cell. To supplement these efforts, artificial metalloenzymes are drawing attention as they combine attractive features of both homogeneous catalysts and enzymes. Herein we show that such hybrid catalysts consisting of a metal cofactor, a cell-penetrating module, and a protein scaffold are taken up into HEK-293T cells where they catalyze the uncaging of a hormone. This bioorthogonal reaction causes the upregulation of a gene circuit, which in turn leads to the expression of a nanoluc-luciferase. Relying on the biotin–streptavidin technology, variation of the biotinylated ruthenium complex: the biotinylated cell-penetrating poly(disulfide) ratio can be combined with point mutations on streptavidin to optimize the catalytic uncaging of an allyl-carbamate-protected thyroid hormone triiodothyronine. These results demonstrate that artificial metalloenzymes offer highly modular tools to perform bioorthogonal catalysis in live HEK cells.
Metal: RuHost protein: Streptavidin (Sav)Anchoring strategy: SupramolecularReaction: DeallylationMax TON: 33ee: ---PDB: ---Notes: ---
An Evolutionary Path to Altered Cofactor Specificity in a Metalloenzyme
Nat. Commun. 2020, 11, 10.1038/s41467-020-16478-0
AbstractAlmost half of all enzymes utilize a metal cofactor. However, the features that dictate the metal utilized by metalloenzymes are poorly understood, limiting our ability to manipulate these enzymes for industrial and health-associated applications. The ubiquitous iron/manganese superoxide dismutase (SOD) family exemplifies this deficit, as the specific metal used by any family member cannot be predicted. Biochemical, structural and paramagnetic analysis of two evolutionarily related SODs with different metal specificity produced by the pathogenic bacterium Staphylococcus aureus identifies two positions that control metal specificity. These residues make no direct contacts with the metal-coordinating ligands but control the metal’s redox properties, demonstrating that subtle architectural changes can dramatically alter metal utilization. Introducing these mutations into S. aureus alters the ability of the bacterium to resist superoxide stress when metal starved by the host, revealing that small changes in metal-dependent activity can drive the evolution of metalloenzymes with new cofactor specificity.
Ligand type: Amino acidHost protein: Superoxide dismutase (SOD)Anchoring strategy: DativeReaction: Metal substitutionMax TON: ---ee: ---PDB: ---Notes: PDB: 6EX3, 6EX4, 6EX5, 6QV8, 6QV9
Construction and In Vivo Assembly of a Catalytically Proficient and Hyperthermostable De Novo Enzyme
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.
Metal: FeLigand type: PorphyrinHost protein: C45 (c-type cytochrome maquette)Anchoring strategy: SupramolecularReaction: OxidationMax TON: ---ee: ---PDB: ---Notes: Oxidation of 2,2′-azino-bis(3-ethylbenzothiazo-line-6-sulfonic acid (ABTS)
Engineering a Dirhodium Artificial Metalloenzyme for Selective Olefin Cyclopropanation
Nat. Commun. 2015, 6, 10.1038/ncomms8789
Artificial metalloenzymes (ArMs) formed by incorporating synthetic metal catalysts into protein scaffolds have the potential to impart to chemical reactions selectivity that would be difficult to achieve using metal catalysts alone. In this work, we covalently link an alkyne-substituted dirhodium catalyst to a prolyl oligopeptidase containing a genetically encoded L-4-azidophenylalanine residue to create an ArM that catalyses olefin cyclopropanation. Scaffold mutagenesis is then used to improve the enantioselectivity of this reaction, and cyclopropanation of a range of styrenes and donor–acceptor carbene precursors were accepted. The ArM reduces the formation of byproducts, including those resulting from the reaction of dirhodium–carbene intermediates with water. This shows that an ArM can improve the substrate specificity of a catalyst and, for the first time, the water tolerance of a metal-catalysed reaction. Given the diversity of reactions catalysed by dirhodium complexes, we anticipate that dirhodium ArMs will provide many unique opportunities for selective catalysis.
Metal: RhLigand type: Poly-carboxylic acidHost protein: Prolyl oligopeptidase (POP)Anchoring strategy: CovalentOptimization: Chemical & geneticReaction: CyclopropanationMax TON: 74ee: 92PDB: ---Notes: ---