Artificial metalloenzyme explained

An Artificial Metalloenzyme (ArM) is a designer metalloprotein, not found in nature, which can catalyze desired chemical reactions.[1] [2] Despite fitting into classical enzyme categories, ArMs also have potential in new-to-nature chemical reactivity like catalysing Suzuki coupling,[3] Metathesis[4] etc., which were never reported among natural enzymatic reactions. ArMs have two main components: a protein scaffold and an artificial catalytic moiety, which, in this case, features a metal center. This class of designer biocatalysts is unique because of the potential to improve the catalytic performance through chemogenetic optimization, a parallel improvement of both the direct metal surrounding (first coordination sphere) and the protein scaffold (second coordination sphere).The second coordination sphere (protein scaffold) is easily evolvable and, in the case of ArMs, responsible for very high (stereo)selectivity. With the progress in organometallic synthesis and protein engineering, more and more new kind of design of ArMs were developed, showing promising future in both academia and industrial aspects.[5]

In 2018, one-half of the Nobel Prize in Chemistry was awarded to Frances H. Arnold "for the directed evolution of enzymes", who elegantly evolved artificial metalloenzymes to realize efficient and highly selective new-to-nature chemical reactions in vitro and in vivo.

History

Dated back to 1956, the first protein modified transition metal catalyst was documented.[6] The Palladium(II) salt was absorbed onto silk fibroin fiber, reduced by hydrogen to get the first reported ArM, which can catalyze asymmetric hydrogenation. This work was not reproducible, but it is considered to be the first work in the field of artificial metalloenzymes. At that time, the major challenge that blocked further studies was underdeveloped protein production and purification technology. The first attempt to anchor an abiotic metal center onto a protein was reported by Whitesides et al. using biotin-avidin interaction, making an artificial hydrogenase.[7] The presence of avidin can significantly increase the catalytic capacity of Rhodium(I) cofactor in aqueous phosphate buffer. Another pioneering work was conducted by Kaiser et al. where carboxypeptidase A (CPA) was repurposed into an oxidase by substituting Zn(II) center by Cu(II), for the oxidation of ascorbic acid.[8]

The real potential of ArMs was unleashed when recombinant protein production was developed, namely in 1997 Distefano and Davies reported a scaffold modification of a recombinant adipocyte lipid-binding protein (ALBP) with iodoacetamido-1,10-phenanthroline coordinating Cu(II) for the stereoselective hydrolysis of racemic esters.[9]

Formation

Abiotic cofactor anchoring

Four strategies have been used to assemble ArMs:

  1. Covalent immobilization of a metal-containing catalytic moiety by an irreversible reaction with the protein;
  2. Supramolecular interactions between a protein and a high-affinity substrate could be used to anchor a metal cofactor;
  3. The metal substitution in a natural metalloenzyme can result in a novel catalytic activity to the protein. The metal could be part of a prosthetic group (e.g., heme) or bound to amino acids;
  4. Amino acids with Lewis-basic properties in a hydrophobic pocket could interact with coordinatively unsaturated metal center.

These four strategies led to a great progress in the field of artificial metalloenzymes since the beginning of the 21st century, unlocking exceptional selectivity for new-to-nature reactions.

Covalent

With the development of bioconjugation technology, there are plenty of strategies to covalently bind an artificial metallocofactor onto a protein scaffold:

Supramolecular

Streptavidin or avidin in combination with biotinylated artificial metal cofactors is the most commonly used supramolecular strategy to make ArMs.[15] In the early example from Ward et al. shown below, the ligand of Ru(I) complex was covalently linked to biotin and than the whole complex was anchored to streptavidin thanks to a specific and strong biotin-streptavidin interaction.[16] 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. 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.[17]

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.[18] 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;[19] [20] and Fe heme complex which catalyzed cyclopropanation enantioselectively.[21]

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.[22] 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)[23] 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)[24] and 8-hydroxyquinoline (2-amino-3-(8-hydroxyquinolin-3-yl)propanoic acid)[25] that can selectively coordinate different metals. Combining protein scaffolds featuring chelating ncAAs with different metals yields exceptionally selective artificial metalloenzymes with various application potentials.[26] 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,[27] carbene insertion,[28] and aminohydroxylation[29] 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),[30] and Huang et al. repurposed non-heme Fe-dependent enzymes to catalyze an abiological radical-relay azidation[31] and radical fluorination.[32] [33]

Function

So far, ArMs can catalyze planty of chemical reactions, such as: allylic alkylation, allylic amination, aldol reaction, alcohol oxidation, C-H activation,[34] click reaction,[35] catechol oxidation, reduction, cyclopropanation,[36] Diels-Alder reaction,[37] epoxidation, epoxide ring opening, Friedel-Crafts alkylation,[38] hydrogenation, hydroformylation, Heck reaction, Metathesis, Michael addition, nitrite reduction, NO reduction, Suzuki reaction, Si-H insertion,[39] polymerization (atom transfer radical polymerization),[40] atom transfer radical cyclization (ATRC) and radical fluorination.

Notes and References

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  2. Leurs M, Dorn B, Wilhelm S, Manisegaran M, Tiller JC . Multicore Artificial Metalloenzymes Derived from Acylated Proteins as Catalysts for the Enantioselective Dihydroxylation and Epoxidation of Styrene Derivatives . Chemistry: A European Journal . 24 . 42 . 10859–10867 . July 2018 . 29808506 . 10.1002/chem.201802185 .
  3. Chatterjee A, Mallin H, Klehr J, Vallapurackal J, Finke AD, Vera L, Marsh M, Ward TR . An enantioselective artificial Suzukiase based on the biotin-streptavidin technology . Chemical Science . 7 . 1 . 673–677 . January 2016 . 29896353 . 5953008 . 10.1039/C5SC03116H .
  4. Jeschek M, Reuter R, Heinisch T, Trindler C, Klehr J, Panke S, Ward TR . Directed evolution of artificial metalloenzymes for in vivo metathesis . Nature . 537 . 7622 . 661–665 . September 2016 . 27571282 . 10.1038/nature19114 . 2016Natur.537..661J . 205250261 .
  5. Schwizer F, Okamoto Y, Heinisch T, Gu Y, Pellizzoni MM, Lebrun V, Reuter R, Köhler V, Lewis JC, Ward TR . Artificial Metalloenzymes: Reaction Scope and Optimization Strategies . Chemical Reviews . 118 . 1 . 142–231 . January 2018 . 28714313 . 10.1021/acs.chemrev.7b00014 .
  6. Akabori S, Sakurai S, Izumi Y, Fujii Y . August 1956. An Asymmetric Catalyst. Nature. 178. 4528. 323–324. 10.1038/178323b0. 0028-0836. 1956Natur.178..323A. 13358737. 4221816.
  7. Wilson ME, Whitesides GM. January 1978. Conversion of a protein to a homogeneous asymmetric hydrogenation catalyst by site-specific modification with a diphosphinerhodium(I) moiety. Journal of the American Chemical Society. 100. 1. 306–307. 10.1021/ja00469a064. 0002-7863.
  8. Yamamura . Kazuo . Kaiser . Emil Thomas . 1976-01-01 . Studies on the oxidase activity of copper(II) carboxypeptidase A . Journal of the Chemical Society, Chemical Communications . en . 20 . 830–831 . 10.1039/C39760000830 . 0022-4936.
  9. Davies . Ronald R. . Distefano . Mark D. . 1997-12-01 . A Semisynthetic Metalloenzyme Based on a Protein Cavity That Catalyzes the Enantioselective Hydrolysis of Ester and Amide Substrates . Journal of the American Chemical Society . 119 . 48 . 11643–11652 . 10.1021/ja970820k . 0002-7863.
  10. Onoda A, Kihara Y, Fukumoto K, Sano Y, Hayashi T . August 2014 . Photoinduced Hydrogen Evolution Catalyzed by a Synthetic Diiron Dithiolate Complex Embedded within a Protein Matrix. ACS Catalysis . 4. 8. 2645–2648. 10.1021/cs500392e .
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  12. Platis IE, Ermácora MR, Fox RO . Oxidative polypeptide cleavage mediated by EDTA-Fe covalently linked to cysteine residues . Biochemistry . 32 . 47 . 12761–7 . November 1993 . 8251497 . 10.1021/bi00210a027 .
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  20. Zhou . Zhi . Roelfes . Gerard . 2021-07-13 . Synergistic Catalysis of Tandem Michael Addition/Enantioselective Protonation Reactions by an Artificial Enzyme . ACS Catalysis . 11 . 15 . 9366–9369 . 10.1021/acscatal.1c02298 . 34386272 . 8353628 . 2155-5435.
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