Artificial Metalloenzymes Research Articles

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.

Synthetic methods: part (ii) oxidation and reduction methods

This Report highlights key advances in some of the most commonly used oxidation and reduction reactions, focusing on the literature from 2011. The structure of this review follows that of the previous years Report by Asghar and Lewis. (S. F. Asghar and S. E. Lewis, Annu. Rep. Prog. Chem., Sect. B, 2011, 107, 34, ref 1). In the field of oxidation, a number of prominent advances have emerged. Notably, Shi and co-workers continue to build on their work in the deamination field through their detailed studies on the regioselectivity of the Cu(I)-catalysed deamination of conjugated dienes (Y. Shi et al. J. Am. Chem. Soc., 2011, ref 36). They reveal that the regioselectivity is governed by the the choice of Cu(I) catalyst and the substituents on the diene substrate. There have also been significant advances in the area of C–H oxidation, with Stambuli showing the stereoselectivity of cis-vinylsilane allylic acetoxylation to be dependent upon the nature of the oxidant used (Stambuli et al. J. Am. Chem. Soc., 2011, ref 66). In the field of reduction chemistry, the Ward group developed an artificial metalloenzyme, where an iridium-catalyst was incorporated into a chiral enzyme for the asymmetric transfer of hydrogen for the production of enantiopure amines (Ward et al. Angew. Chem. Int. Ed., 2011, ref 129). Meanwhile, Strotman and co-authors have demonstrated the first intramolecular reductive amination between dialkyl ketones and aliphatic amines. (Strotman et al. J. Am. Chem. Soc., 2011, ref 121).

Artificial Metalloenzymes as Catalysts in Stereoselective Diels–Alder Reactions

Numerous enzymes are useful catalysts in synthetic organic chemistry, but they cannot catalyze the myriad transition-metal-mediated transformations customary in daily chemical work. For this reason the concept of directed evolution of hybrid catalysts was proposed some time ago. A synthetic ligand/transition-metal moiety is anchored covalently or non-covalently to a host protein, thereby generating a single artificial metalloenzyme which can then be optimized by molecular biological methods. In the quest to construct an appropriate experimental platform for asymmetric Diels-Alder reactions amenable to this Darwinian approach to catalysis, specifically those not currently possible using traditional chiral transition-metal catalysts, two strategies have been developed which are reviewed here. One concerns the supramolecular anchoring of a Cu(II)-phthalocyanine complex to serum albumins; the other is based on the design of a Cu(II)-specific binding site in a thermostable protein host (tHisF), leading to 46-98% ee in a model Diels-Alder reaction. This sets the stage for genetic fine-tuning using the methods of directed evolution.

Enantioselective Artificial Metalloenzymes by Creation of a Novel Active Site at the Protein Dimer Interface

A game of two halves: Artificial metalloenzymes are generated by forming a novel active site on the dimer interface of the transcription factor LmrR. Two copper centers are incorporated by binding to ligands in each half of the dimer. With this system up to 97 % ee was obtained in the benchmark CuII catalyzed Diels–Alder reaction (see scheme). Detailed facts of importance to specialist readers are published as ”Supporting Information”. Such documents are peer-reviewed, but not copy-edited or typeset. They are made available as submitted by the authors. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Enantioselective Artificial Metalloenzymes by Creation of a Novel Active Site at the Protein Dimer Interface

Enantioselective Artificial Metalloenzymes by Creation of a Novel Active Site at the Protein Dimer Interface

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.

ChemInform Abstract: Artificial Metalloenzymes for Olefin Metathesis Based on the Biotin‐(strept)avidin Technology.

AbstractIncorporation of a biotinylated Hoveyda—Grubbs catalyst within (strept)avidin affords artificial metalloenzymes for the ring‐closing metathesis of diallylamine (I) in aqueous solution.

Identification of two-histidines one-carboxylate binding motifs in proteins amenable to facial coordination to metals

Among natural metalloenzymes, the facial two-histidines one-carboxylate binding motif (FTM) is a widely represented first coordination sphere motif present in the active site of a variety of metalloenzymes. A PDB search revealed a total of 1685 structures bearing such FTMs bound to a metal. Sixty statistically representative FTMs were selected and used as template for the identification of structurally characterized proteins bearing these three amino acids in a propitious environment for binding to a transition metal. This geometrical superposition search, carried out using the STAMPS software, returned 2320 hits. While most consisted of either apo-FTMs or bore strong sequence homology to known FTMs, seven such structures lying within a cavity were identified as novel and viable scaffolds for the creation of artificial metalloenzymes bearing an FTM.

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.

Enantioselective transfer hydrogenation of ketone catalysed by artificial metalloenzymes derived from bovine β-lactoglobulin

Artificial metalloproteins resulting from the embedding of half-sandwich Ru(II)/Rh(III) fatty acid derivatives within β-lactoglobulin catalysed the asymmetric transfer hydrogenation of trifluoroacetophenone with modest to good conversions and fair ee's.

Editorial: Onwards and Upwards-The Future Is Bright for Chemistry!

Editorial: Onwards and Upwards-The Future Is Bright for Chemistry!

Incorporation of Manganese Complexes into Xylanase: New Artificial Metalloenzymes for Enantioselective Epoxidation

Here we report the best artificial metalloenzyme to date for the selective oxidation of aromatic alkenes; it was obtained by noncovalent insertion of Mn(III)-meso-tetrakis(p-carboxyphenyl)porphyrin [Mn(TpCPP), 1-Mn] into a host protein, xylanase 10A from Streptomyces lividans (Xln10A). Two metallic complexes-N,N'-ethylene bis(2-hydroxybenzylimine)-5,5'-dicarboxylic acid Mn(III) [(Mn-salen), 2-Mn] and 1-Mn-were associated with Xln10A, and the two hybrid biocatalysts were characterised by UV-visible spectroscopy, circular dichroism and molecular modelling. Only the artificial metalloenzyme based on 1-Mn and Xln10A was studied for its catalytic properties in the oxidation of various substituted styrene derivatives by KHSO(5): after optimisation, the 1-Mn-Xln10A artificial metalloenzyme was able to catalyse the oxidation of para-methoxystyrene by KHSO(5) with a 16 % yield and the best enantioselectivity (80 % in favour of the R isomer) ever reported for an artificial metalloenzyme.

ChemInform Abstract: Merging the Best of Two Worlds: Artificial Metalloenzymes for Enantioselective Catalysis

AbstractReview: 61 refs.

OsO4⋅Streptavidin: A Tunable Hybrid Catalyst for the Enantioselective cis‐Dihydroxylation of Olefins

Enzymatic and homogeneous catalysis have evolved independently to address the challenges in the synthesis of enantiopure products. With the aim of complementing these fields, artificial metalloenzymes, which combine the structural diversity of biocatalysts with the wealth of metal-catalyzed reactions, have attracted increasing attention. [1] In homogeneous catalysis the cis-selective, OsO4-dependent asymmetric dihydroxylation (AD) of olefins ranks among the most powerful methods for the synthesis of vicinal diols. Ligands for homogeneous catalysis have been largely developed by Sharpless and co-workers, and are, with few exceptions, almost exclusively based on quinidine or quinine derivatives. [2] Although most classes of prochiral olefins are dihydroxylated with good activity and selectivity, the cissubstituted olefins are problematic. Nature relies on nonheme iron dioxygenases such as naphthalene dioxygenase (NDO) to perform a related reaction. These enzymes display broad substrate scope. [3] It is believed that both the OsO4- and NDO-catalyzed dihydroxylations proceed by an outer sphere [3+2] mechanism in which the substrate is not bound to the

ChemInform Abstract: Bioinspired Catalyst Design and Artificial Metalloenzymes

AbstractReview: more than 200 refs.

ChemInform Abstract: Enzymatic Functionalization of Carbon—Hydrogen Bonds

AbstractReview: 115 refs.

Bioinspired Catalyst Design and Artificial Metalloenzymes

Many bioinspired transition-metal catalysts have been developed over the recent years. In this review the progress in the design and application of ligand systems based on peptides and DNA and the development of artificial metalloenzymes are reviewed with a particular emphasis on the combination of phosphane ligands with powerful molecular recognition and shape selectivity of biomolecules. The various approaches for the assembly of these catalytic systems will be highlighted, and the possibilities that the use of the building blocks of Nature provide for catalyst optimisation strategies are discussed.

Burkavidin: A novel secreted biotin-binding protein from the human pathogen Burkholderia pseudomallei

The avidin–biotin technology has many applications, including molecular detection; immobilization; protein purification; construction of supramolecular assemblies and artificial metalloenzymes. Here we present the recombinant expression of novel biotin-binding proteins from bacteria and the purification and characterization of a secreted burkavidin from the human pathogen Burkholderia pseudomallei. Expression of the native burkavidin in Escherichia coli led to periplasmic secretion and formation of a biotin-binding, thermostable, tetrameric protein containing an intra-monomeric disulphide bond. Burkavidin showed one main species as measured by isoelectric focusing, with lower isoelectric point (pI) than streptavidin. To exemplify the potential use of burkavidin in biotechnology, an artificial metalloenzyme was generated using this novel protein-scaffold and shown to exhibit enantioselectivity in a rhodium-catalysed hydrogenation reaction.

Merging the best of two worlds: artificial metalloenzymes for enantioselective catalysis

Artificial metalloenzymes result from combining a catalytically active organometallic moiety with a macromolecular host. The resulting hybrid catalysts combine attractive features of both homogeneous and enzymatic systems. Herein we summarize the recent progress in this emerging field and outline the challenges ahead.

Enzymatic functionalization of carbon-hydrogen bonds.

The development of new catalytic methods to functionalize carbon-hydrogen (C-H) bonds continues to progress at a rapid pace due to the significant economic and environmental benefits of these transformations over traditional synthetic methods. In nature, enzymes catalyze regio- and stereoselective C-H bond functionalization using transformations ranging from hydroxylation to hydroalkylation under ambient reaction conditions. The efficiency of these enzymes relative to analogous chemical processes has led to their increased use as biocatalysts in preparative and industrial applications. Furthermore, unlike small molecule catalysts, enzymes can be systematically optimized via directed evolution for a particular application and can be expressed in vivo to augment the biosynthetic capability of living organisms. While a variety of technical challenges must still be overcome for practical application of many enzymes for C-H bond functionalization, continued research on natural enzymes and on novel artificial metalloenzymes will lead to improved synthetic processes for efficient synthesis of complex molecules. In this critical review, we discuss the most prevalent mechanistic strategies used by enzymes to functionalize non-acidic C-H bonds, the application and evolution of these enzymes for chemical synthesis, and a number of potential biosynthetic capabilities uniquely enabled by these powerful catalysts (110 references).