Design Of Artificial Metalloenzymes Research Articles

Oxidation Catalysis by Rationally Designed Artificial Metalloenzymes

AbstractThe principle of enzyme mimics has been raised to its pinnacle by the design of hybrids made from inorganic complexes embedded into biomolecules. The present review focuses on the design of artificial metalloenzymes for oxidation reactions by oxygen transfer reactions, with a special focus on proteins anchoring inorganic complexes or metal ions via supramolecular interactions. Such reactions are of great interest for the organic synthesis of building blocks. In the first part, following an overview of the different design of artificial enzymes, the review presents contributions to the rational design of efficient hybrid biocatalysts via supramolecular host/guest approaches, based on the nature of the inorganic complex and the nature of the protein, with special attention to the substrate binding. In the second part, the original purpose of artificial metalloenzymes has been twisted to enable the observation of transient intermediates, to decipher metal‐based oxidation mechanisms. The host protein crystals have been used as crystalline molecular‐scale vessels, within which inorganic catalytic reactions have been followed, thanks to X‐ray crystallography. These hybrids should be an alternative to enzymes for sustainable chemistry.

Unravelling novel synergies between organometallic and biological partners: a quantum mechanics/molecular mechanics study of an artificial metalloenzyme.

In recent years, the design of artificial metalloenzymes obtained by the insertion of homogeneous catalysts into biological macromolecules has become a major field of research. These hybrids, and the corresponding X-ray structures of several of them, are offering opportunities to better understand the synergy between organometallic and biological subsystems. In this work, we investigate the resting state and activation process of a hybrid inspired by an oxidative haemoenzyme but presenting an unexpected reactivity and structural features. An extensive series of quantum mechanics/molecular mechanics calculations show that the resting state and the activation processes of the novel enzyme differ from naturally occurring haemoenzymes in terms of the electronic state of the metal, participation of the first coordination sphere of the metal and the dynamic process. This study presents novel insights into the sensitivity of the association between organometallic and biological partners and illustrates the molecular challenge that represents the design of efficient enzymes based on this strategy.

Crystal Structure of Apo‐ and Metalated Thiolate containing RNase S as Structural Basis for the Design of Artificial Metalloenzymes by Peptide‐Protein Complementation

AbstractRibonuclease S (RNase S) is a versatile model system to introduce non‐canonical amino acids into a protein scaffold by self‐assembly of the protein complex from S‐protein and S‐peptide. We have introduced two cysteines into the S‐peptide replacing lysine‐7 and glutamine‐11 of the α‐helical region of this peptide. The crystal structure of this variant was determined in the apo state and in complex with HgII ions. These structures serve as a template for the construction of artificial hybrid catalysts based on RNase S, e.g. for phosphanyl amino acids complexing transition metal ions for organometallic catalysis.

ChemInform Abstract: Artificial Metalloenzymes Constructed from Hierarchically‐Assembled Proteins

AbstractReview: 121 refs.

Artificial Metalloenzymes Constructed From Hierarchically‐Assembled Proteins

The design of artificial metalloenzymes has become an important topic in biological chemistry and inorganic chemistry due to the potential applications of artificial metalloenzymes in nanoscience and biotechnology. One of the general methods used to produce artificially metalloenzymes involves the encapsulation of non-natural metal cofactors within protein scaffolds. This method has been used in the construction of small artificial metalloproteins with high activity and selectivity. However, the important roles of protein assemblies have not yet been systematically investigated in this field, even though natural enzymatic systems employ protein assemblies as molecular scaffolds for elaborate enzymatic reactions. In recent years, the above-mentioned general strategy has been applied to functionalize protein assemblies such as protein cages and protein crystals. These assembled structures form confined interior environments, which can be used to accommodate metal complex catalysts and to prepare metal nanoparticles. The development of artificial metalloenzymes with hierarchically-assembled proteins would enable us to provide powerful tools for industrial and biological applications. In this Focus Review, we discuss the most significant recent research in this field as well as future directions.

Catalytic Mechanism in Artificial Metalloenzyme: QM/MM Study of Phenylacetylene Polymerization by Rhodium Complex Encapsulated in apo-Ferritin

Artificial metalloenzyme, composed of metal complex(es) and a host protein, is a promising way to mimic enzyme catalytic functions or develop novel enzyme-like catalysis. However, it is highly challenging to unveil the active site and exact reaction mechanism inside artificial metalloenzyme, which is the bottleneck in its rational design. We present a QM/MM study of the complicated reaction mechanism for the recently developed artificial metalloenzyme system, (Rh(nbd)·apo-Fr) (nbd = norbornadiene), which is composed of a rhodium complex [Rh(nbd)Cl](2) and the recombinant horse L-chain apo-Ferritin. We found that binding sites suggested by the X-ray crystal structure, i.e., sites A, B, and C, are only precursors/intermediates, not true active sites for polymerization of phenylacetylene (PA). A new hydrophobic site, which we name D, is suggested to be the most plausible active site for polymerization. Active site D is generated after coordination of first monomer PA by extrusion of the Rh(I)(PA) complex to a hydrophobic pocket near site B. Polymerization occurs in site D via a Rh(I)-insertion mechanism. A specific "hydrophobic region" composed by the hydrophobic active site D, the nonpolar 4-fold channel, and other hydrophobic residues nearby is found to facilitate accumulation, coordination, and insertion of PA for polymerization. Our results also demonstrate that the hydrophobic active site D can retain the native regio- and stereoselectivity of the Rh-catalyzed polymerization of PA without protein. This study highlights the importance of theoretical study in mechanistic elucidation and rational design of artificial metalloenzymes, indicating that even with X-ray crystal structures at hand we may still be far from fully understanding the active site and catalytic mechanism of artificial metalloenzymes.

The structure of the periplasmic nickel-binding protein NikA provides insights for artificial metalloenzyme design

Understanding the interaction of a protein with a relevant ligand is crucial for the design of an artificial metalloenzyme. Our own interest is focused on the synthesis of artificial monooxygenases. In an initial effort, we have used the periplasmic nickel-binding protein NikA from Escherichia coli and iron complexes in which N(2)Py(2) ligands (where Py is pyridine) have been varied in terms of charge, aromaticity, and size. Six "NikA/iron complex" hybrids have been characterized by X-ray crystallography, and their interactions and solution properties have been studied. The hybrids are stable as indicated by their K (d) values, which are all in the micromolar range. The X-ray structures show that the ligands interact with NikA through salt bridges with arginine residues and π-stacking with a tryptophan residue. We have further characterized these interactions using quantum mechanical calculations and determined that weak CH/π hydrogen bonds finely modulate the stability differences between hybrids. We emphasize the important role of the tryptophan residues. Thus, our study aims at the complete characterization of the factors that condition the interaction of an artificial ligand and a protein and their implications for catalysis. Besides its potential usefulness in the synthesis of artificial monooxygenases, our approach should be generally applicable in the field of artificial metalloenzymes.

Semi-synthesis of an artificial scandium(iii) enzyme with a β-helical bio-nanotube

We have succeeded in preparing semi-synthesized proteins bound to Sc(3+) ion which can promote an epoxide ring-opening reaction. The Sc(3+) binding site was created on the surface of [(gp5βf)(3)](2) (N. Yokoi et al., Small, 2010, 6, 1873) by introducing a cysteine residue for conjugation of a bpy moiety using a thiol-maleimide coupling reaction. Three cysteine mutants [(gp5βf_X)(3)](2) (X = G18C, L47C, N51C) were prepared to introduce a bpy in different positions because it had been reported that Sc(3+) ion can serve as a Lewis-acid catalyst for an epoxide ring-opening reaction upon binding of epoxide to bpy and two -ROH groups. G18C_bpy with Sc(3+) can accelerate the rate of catalysis of the epoxide ring-opening reaction and has the highest rate of conversion among the three mutants. The value is more than 20 times higher than that of the mixtures of [(gp5βf)(3)](2)/2,2'-bipyridine and L-threonine/2,2'-bipyridine. The elevated activity was obtained by the cooperative effect of stabilizing the Sc(3+) coordination and accumulation of substrates on the protein surface. Thus, we expect that the semi-synthetic approach can provide insights into new rational design of artificial metalloenzymes.

Design of a Functional Nitric Oxide Reductase within a Myoglobin Scaffold

A review. In recent years, significant progress has been made in the computational design of functional artificial enzymes. When combined with directed evolution protocols, their efficiencies are comparable to those obtained with catalytic antibodies. In comparison, the in silico creation of artificial metalloenzymes remains challenging. This could be due to the difficulty in computing both the transition states for metal-catalyzed reactions and the corresponding entatic state for a metalloenzyme. In a recent study, structurally characterized a functional bacterial nitric oxide reductase within a myoglobin scaffold has been rationally designed. This ground-breaking study by Lu and coworkers has opened the way towards the rational design of artificial metalloenzymes for more challenging reactions. For the artificial nitric oxide reductase, several issues deserve further scrutiny: catalytic efficiency as well as detailed reaction mechanism. In a broader perspective, the use of more elaborate computational algorithms, combined with efficient directed-evolution protocols should enable the creation and optimization of highly versatile artificial metalloenzymes in a variety of protein folds. [on SciFinder(R)]

Design of artificial metalloenzymes using non-covalent insertion of a metal complex into a protein scaffold

Construction of artificial metalloenzymes is one of the most attractive targets in the field of inorganic and catalytic chemistry, since they show remarkable chemoselectivity and reactivity in aqueous media. For the purpose, covalent modification of protein and cofactors have usually been utilized to attach a metal complex(es) to a protein scaffold. This article focuses on non-covalent insertion of metal complexes into protein environments. The discussion includes the screening of stable metal complex/protein composites, crystal structures, molecular design for regulating enantioselectivity of the target catalytic reactions. Our recent results show that the non-covalent conjugation will provide us a new way in semi-synthesis of artificial metalloenzymes.

Crystallographic and Spectroscopic Evidence for High Affinity Binding of FeEDTA(H2O)-to the Periplasmic Nickel Transporter NikA

Because nickel is both essential and toxic to a great variety of organisms, its detection and transport is highly regulated. In Escherichia coli and other related Gram-negative bacteria, high affinity nickel transport depends on proteins expressed by the nik operon. A central actor of this process is the periplasmic NikA transport protein. A previous structural report has proposed that nickel binds to NikA as a pentahydrate species. However, both stereochemical considerations and X-ray absorption spectroscopic results are incompatible with that interpretation. Here, we report the 1.8 A resolution structure of NikA and show that it binds FeEDTA(H2O)- with very high affinity. In addition, we provide crystallographic evidence that a metal-EDTA complex was also bound to the previously reported NikA structure. Our observations strongly suggest that nickel transport in E. coli requires the binding of this metal ion to a metallophore that bears significant resemblance to EDTA. They also provide a basis for the potential use of NikA in the bioremediation of toxic transition metals and the design of artificial metalloenzymes.

Design of artificial metalloenzymes

Homogeneous and enzymatic catalysis offer complementary means to generate enantiomerically pure compounds. For this reason, in a biomimetic spirit, efforts are currently under way in different groups to design artificial enzymes. Two complementary strategies are possible to incorporate active organometallic catalyst precursors into a protein environment. The first strategy utilizes covalent anchoring of the organometallic complexes into the protein environment. The second strategy relies on the use of non-covalent incorporation of the organometallic precursor into the protein. In this review, attention is focused on the use of semisynthetic enzymes to produce efficient enantioselective hybrid catalysts for a given reaction. This article also includes our recent research results and implications in developing the biotin–avidin technology to localize the biotinylated organometallic catalyst precursor within a well-defined protein environment. Copyright © 2005 John Wiley & Sons, Ltd.