File:Cu(II)-bipyridine-papain complex.webm|305x305px|An artificial oxidase based on Cu(II)-bipyridine complex linked to the cysteine in the active site of adipocyte lipid binding protein (PDB: 1A18). Artificial ligand showed in red (copper not shown).|thumb
Abiotic cofactor anchoring Four strategies have been used to assemble ArMs: Cys-
α-haloketone, Cys-benzylhalide chemistry and
disulfide formation, • post-translational
bioorthogonal modification based on Amber stop codon suppression (e.g., Click chemistry) • enzyme active site modification (e.g., covalent bond formation between lipase and lipase inhibitor).
Supramolecular Streptavidin or
avidin in combination with
biotinylated artificial metal cofactors is the most commonly used supramolecular strategy to make ArMs. In the early example from Ward
et al. shown below, the ligand of Ru(I) complex was covalently linked to biotin and then the whole complex was anchored to
streptavidin thanks to a specific and strong biotin-streptavidin interaction. The formed ArM can catalyze the reduction of
prochiral ketones. Taking advantages of protein evolvability, different mutants of streptavidin can achieve different stereoselectivity. Throughout the years, many streptavidin-based enzymes were developed, enabling catalysis of very complex transformations in water, under ambient conditions. File:Ru-Sav-complex.webm|305x305px|An ArM using biotin-streptavidin interaction to anchor artificial metal cofactor (PDB: 2QCB)|thumb|left Besides biotin-streptavidin based ArMs, another important example of using supramolecular iassembly strategy is
antigen-
antibody recognition. First reported in 1989 by Lerner
et al.., a monoclonal antibody-based ArM is raised to
hydrolyze specific peptide. Another interesting scaffold used as a platform for supramolecularly assembled ArMs are
multidrug resistance regulators (MDRs), particularly a PadR family of proteins without native catalytic activity, whose function in nature is the recognition of foreign agents and to activate subsequent cellular response. Among them, Lactococcal multidrug resistance regulator (LmrR) was mainly used to create ArMs, using different strategies, including the supramolecular one. Namely, Roelfes
et al. incorporated Cu(II)
phenanthroline complex in the hydrophobic pocket of LmrR and performed
Friedel-Crafts reaction enantioselectively; and Fe
heme complex which catalyzed
cyclopropanation enantioselectively.
Metal substitution This strategy involves substitution of a native metal center in a metallocofactor, by another metal, that might or might not be already present in living systems. In this way, electronic and steric properties of the catalytic active site are altered compared to the wild-type enzyme, and novel catalytic pathways are unlocked.
Dative The dative anchoring strategy uses natural amino acid residue in the protein scaffold like
His,
Cys,
Glu,
Asp and
Ser to coordinate to a metal center. Like the first example of Pd-fibroin, dative anchoring to natural amino acids is not commonly used nowadays and often resulted in a more ambiguous binding site for metal compared with previous three methods. However, these challenges can be overcome by
in vivo incorporating
metal-chelating non-canonical amino acids (ncAAs) in the protein scaffold. These genetically encoded ncAAs' side chains have chelating moieties, such as
2,2'-bipyridine (3-(2,2'-bipyridin-5-yl)-L-alanine) and
8-hydroxyquinoline (2-amino-3-(8-hydroxyquinolin-3-yl)propanoic acid) that can selectively coordinate different metals. Combining protein scaffolds featuring chelating ncAAs with different metals yields exceptionally selective artificial metalloenzymes with various application potentials. ncAAs are usually incorporated through the means of
Amber stop codon suppression, via the orthogonal translation system (OTS).
Natural Metalloenzymes repurposing In addition to anchoring artificial metal center in the protein scaffold, researchers like
Frances Arnold and Yang Yang focused on changing the native environment of natural metallocofactors. Due to the large
sequence space that can be evolved in natural metalloenzymes, they can be evolved to catalyse non-native transformations. This process is known as enzyme repurposing.
Directed evolution is commonly used to tailor the catalytic capacity and repurpose the enzyme function. Mostly based on native
porphyrin-metallocofactor, Arnold's lab has developed many ArMs catalysing
regioselective and/or
enantioselective transformations, such as Carbon-
Boron bond formation,
carbene insertion, and aminohydroxylation by evolving the sequence context of the corresponding ArMs. As the pioneers of metalloredox
radical biocatalysis, Yang
et al. repurposed
cytochrome P450s to catalyze atom transfer radical cyclization (ATRC), and Huang
et al. repurposed
non-heme Fe-dependent enzymes to catalyze an abiological radical-relay
azidation and radical
fluorination. == Function ==