Maréchal, J.-D.; Ward, T.R.

J. Am. Chem. Soc. 2014, 136, 15676-15683, 10.1021/ja508258t

An artificial imine reductase results upon incorporation of a biotinylated Cp*Ir moiety (Cp* = C5Me5–) within homotetrameric streptavidin (Sav) (referred to as Cp*Ir(Biot-p-L)Cl] ⊂ Sav). Mutation of S112 reveals a marked effect of the Ir/streptavidin ratio on both the saturation kinetics as well as the enantioselectivity for the production of salsolidine. For [Cp*Ir(Biot-p-L)Cl] ⊂ S112A Sav, both the reaction rate and the selectivity (up to 96% ee (R)-salsolidine, kcat 14–4 min–1 vs [Ir], KM 65–370 mM) decrease upon fully saturating all biotin binding sites (the ee varying between 96% ee and 45% ee R). In contrast, for [Cp*Ir(Biot-p-L)Cl] ⊂ S112K Sav, both the rate and the selectivity remain nearly constant upon varying the Ir/streptavidin ratio [up to 78% ee (S)-salsolidine, kcat 2.6 min–1, KM 95 mM]. X-ray analysis complemented with docking studies highlight a marked preference of the S112A and S112K Sav mutants for the SIr and RIr enantiomeric forms of the cofactor, respectively. Combining both docking and saturation kinetic studies led to the formulation of an enantioselection mechanism relying on an “induced lock-and-key” hypothesis: the host protein dictates the configuration of the biotinylated Ir-cofactor which, in turn, by and large determines the enantioselectivity of the imine reductase.

Seelig, B.

Nat. Chem. Biol. 2013, 9, 81-83, 10.1038/nchembio.1138

Engineering functional protein scaffolds capable of carrying out chemical catalysis is a major challenge in enzyme design. Starting from a noncatalytic protein scaffold, we recently generated a new RNA ligase by in vitro directed evolution. This artificial enzyme lost its original fold and adopted an entirely new structure with substantially enhanced conformational dynamics, demonstrating that a primordial fold with suitable flexibility is sufficient to carry out enzymatic function.

Herrick, R.S.

J. Organomet. Chem. 2014, 751, 90-110, 10.1016/j.jorganchem.2013.07.004

Bioorganometallic chemistry is a rapidly growing subfield of organometallic chemistry. One important facet is the study of organometallic•protein complexes that contain a covalent bond between the protein and an organometallic prosthetic group. Structural elucidation of these complexes is being used with increasing frequency to determine exactly where metal binding takes place and to obtain accurate structural information. This review summarizes the structures in this field, highlighting how this information has driven the frontier of this research.

Mahy, J.-P.

FEBS Lett. 1999, 443, 229-234, 10.1016/S0014-5793(98)01703-7

The temperature and pH dependence as well as the selectivity of the peroxidase activity of a complex associating a monoclonal antibody 13G10 with its iron(III)‐α,α,α,β‐meso‐tetrakis(ortho‐carboxyphenyl) porphyrin (Fe(ToCPP)) hapten have been studied and compared to those of Fe(ToCPP) alone. It first appears that the peroxidase activity of the 13G10‐Fe(ToCPP) complex is remarkably thermostable and remains about 5 times higher than that of Fe(ToCPP) alone until at least 80°C. Secondly, this complex is able to use not only H2O2 as oxidant but also a wide range of hydroperoxides such as alkyl, aralkyl and fatty acid hydroperoxides and catalyze their reduction 2–6‐fold faster than Fe(ToCPP) alone. It is also able to catalyze the oxidation by H2O2 of a variety of reducing cosubstrates such as 2,2′‐azinobis(3‐ethylbenzothiazoline‐6‐sulfonic acid) (ABTS), o‐phenylenediamine (OPD), 3,3′,5,5′‐tetramethylbenzidine (TMB) and 3,3′‐dimethoxybenzidine 3–8‐fold faster than Fe(ToCPP) alone, the bicyclic aromatic ABTS and TMB being the best reducing cosubstrates. Finally, a pH dependence study, between pH 4.6 and 7.5, of the oxidation of ABTS by H2O2 in the presence of either 13G10‐Fe(ToCPP) or Fe(ToCPP) shows that K m(H2O2) values vary very similarly for both catalysts, whereas very different variations are found for the k cat values. With Fe(ToCPP) as catalyst the k cat value remains constant around 100 min−1 whereas with the 13G10‐Fe(ToCPP) complex, it increases sharply below pH 5 to reach 540 min−1 at pH 4.6. This could be due to the participation of a carboxylic acid side chain of the antibody protein, as a general acid‐base catalyst, to the heterolytic cleavage of the O‐O bond of H2O2 leading to the highly reactive iron(V)‐oxo intermediate in the peroxidase mechanism. Accordingly, the modification of the carboxylic acid residues of antibody 13G10 by glycinamide leads to a 50% decrease of the peroxidase activity of the 13G10‐Fe(ToCPP) complex.

Kaiser, E.T.

J. Chem. Soc., Chem. Commun. 1976, 830, 10.1039/C39760000830

Copper(II) carboxypeptidase A catalyses the oxidation of ascorbic acid and this reaction is inhibited by α-benzylsuccinate, a known inhibitor of the thiolesterase action of the copper enzyme; the pH dependencies of kcat and kcat/Km are similar near pH 7 to those seen for the peptidase and esterase activities of native carboxypeptidase A.

Anderson, J.L.R.

J. Inorg. Biochem. 2021, 217, 111370, 10.1016/j.jinorgbio.2021.111370

The design and construction of de novo enzymes offer potentially facile routes to exploiting powerful chemistries in robust, expressible and customisable protein frameworks, while providing insight into natural enzyme function. To this end, we have recently demonstrated extensive catalytic promiscuity in a heme-containing de novo protein, C45. The diverse transformations that C45 catalyses include substrate oxidation, dehalogenation and carbon‑carbon bond formation. Here we explore the substrate promiscuity of C45's peroxidase activity, screening the de novo enzyme against a panel of peroxidase and dehaloperoxidase substrates. Consistent with the function of natural peroxidases, C45 exhibits a broad spectrum of substrate activities with selectivity dictated primarily by the redox potential of the substrate, and by extension, the active oxidising species in peroxidase chemistry, compounds I and II. Though the comparison of these redox potentials provides a threshold for determining activity for a given substrate, substrate:protein interactions are also likely to play a significant role in determining electron transfer rates from substrate to heme, affecting the kinetic parameters of the enzyme. We also used biomolecular simulation to screen substrates against a computational model of C45 to identify potential interactions and binding sites. Several sites of interest in close proximity to the heme cofactor were discovered, providing insight into the catalytic workings of C45.

Salmain, M.; Thorimbert, S.

Eur. J. Inorg. Chem. 2017, 2017, 3622-3634, 10.1002/ejic.201700365

Several prochiral NCN‐pincer complexes of palladium(II), with hemilabile ligands and a long aliphatic chain, were synthesized and characterized spectroscopically. In some of the complexes, the presence of two different substituents on the N donor atoms made them stereogenic, so that they were isolated as a mixture of diastereoisomers, which could be differentiated by 1H NMR spectroscopy. Binding of some of these complexes to bovine β‐lactoglobin by insertion within its inner cavity was theoretically investigated by molecular‐docking simulations and was experimentally confirmed by CD spectroscopy. Adjunction of H‐bond donor substituents on the ligand framework gave more‐stable supramolecular protein–complex assemblies. These constructs were shown to catalyze aldol condensation reactions in aqueous media, affording, in some cases, the less‐favorable cis product. Since the corresponding complexes exclusively gave the trans product in the absence of β‐lactoglobulin, this unusual diastereoselectivity was ensued by the second sphere of coordination brought by the protein host.

Roelfes, G.

J. Am. Chem. Soc. 2015, 137, 9796-9799, 10.1021/jacs.5b05790

Supramolecular anchoring of transition metal complexes to a protein scaffold is an attractive approach to the construction of artificial metalloenzymes since this is conveniently achieved by self-assembly. Here, we report a novel design for supramolecular artificial metalloenzymes that exploits the promiscuity of the central hydrophobic cavity of the transcription factor Lactococcal multidrug resistance Regulator (LmrR) as a generic binding site for planar coordination complexes that do not provide specific protein binding interactions. The success of this approach is manifested in the excellent enantioselectivities that are achieved in the Cu(II) catalyzed enantioselective Friedel–Crafts alkylation of indoles.

Okamoto, Y.; Ward, T.R.

Comprehensive Supramolecular Chemistry II 2017, 459-510, 10.1016/B978-0-12-409547-2.12551-X

Artificial metalloenzymes result from the incorporation of an organometallic moiety within a macromolecule. In this article, we review the field of artificial metalloenzymes. These are classified according to the host that accommodates the organometallic cofactor: cyclodextrins (“Cyclodextrin-Based Artificial Enzymes” section), ligands bearing a substrate recognition motif (“Artificial Enzymes With Ligands Bearing Substrate Recognition Motifs” section), supramolecular cages (“Cage Molecules as Artificial Enzymes” section), nucleic acids (“DNA-Based Artificial Metalloenzymes” section), and proteins (“Protein-Based Artificial Enzymes” section). Both dative and supramolecular anchoring strategies are reviewed.

Duhme-Klair, A.K.

Dalton Trans. 2009, 10141, 10.1039/b915776j

This perspective illustrates the principles and applications of molecular recognition directed binding of transition metal complexes to proteins. After a brief introduction into non-covalent interactions and the importance of complementarity, the focus of the first part is on biological systems that rely on non-covalent forces for metal complex binding, such as proteins involved in bacterial iron uptake and the oxygen-storage protein myoglobin. The second part of the perspective will illustrate how the replacement of native with non-native metal-centres can give rise to artificial metalloenzymes with novel catalytic properties. Subsequently, examples of spectroscopic probes that exploit the characteristic photophysical properties of metal-complexes for the non-covalent labelling, visualisation and investigation of proteins will be described. Finally, the use of kinetically inert metal complexes as scaffolds in drug design will be discussed and it will be highlighted how the binding of metal ions or organometallic fragments to existing drugs or drug candidates can improve their activity or even alter their mode of action.

Song, W.J.

Chem. Sci. 2021, 12, 5091-5101, 10.1039/d0sc06823c

Directed evolution has provided us with great opportunities and prospects in the synthesis of tailor-made proteins. It, however, often requires at least mid to high throughput screening, necessitating more effective strategies for laboratory evolution. We herein demonstrate that protein symmetry can be a versatile criterion for searching for promising hotspots for the directed evolution of de novo oligomeric enzymes. The randomization of symmetry-related residues located at the rotational axes of artificial metallo-β-lactamase yields drastic effects on catalytic activities, whereas that of non-symmetry-related, yet, proximal residues to the active site results in negligible perturbations. Structural and biochemical analysis of the positive hits indicates that seemingly trivial mutations at symmetry-related spots yield significant alterations in overall structures, metal-coordination geometry, and chemical environments of active sites. Our work implicates that numerous artificially designed and natural oligomeric proteins might have evolutionary advantages of propagating beneficial mutations using their global symmetry.

Roelfes, G.

Nat. Catal. 2020, 3, 289-294, 10.1038/s41929-019-0420-6

Artificial enzymes, which are hybrids of proteins with abiological catalytic groups, have emerged as a powerful approach towards the creation of enzymes for new-to-nature reactions. Typically, only a single abiological catalytic moiety is incorporated. Here we introduce a design of an artificial enzyme that comprises two different abiological catalytic moieties and show that these can act synergistically to achieve high activity and enantioselectivity (up to >99% e.e.) in the catalysed Michael addition reaction. The design is based on the lactococcal multidrug resistance regulator as the protein scaffold and combines a genetically encoded unnatural p-aminophenylalanine residue (which activates an enal through iminium ion formation) and a supramolecularly bound Lewis acidic Cu(ii) complex (which activates the Michael donor by enolization and delivers it to one preferred prochiral face of the activated enal). This study demonstrates that synergistic combination of abiological catalytic groups is a robust way to achieve catalysis that is normally outside of the realm of artificial enzymes.

Filice, M.

Chem. Commun. 2015, 51, 9324-9327, 10.1039/C5CC02450A

A solid-phase strategy using lipase as a biomolecular scaffold to produce a large amount of Cu2+-metalloenzyme is proposed here. The application of this protocol on different 3D cavities of the enzyme allows creating a heterogeneous artificial metallolipase showing chimeric catalytic activity. The artificial catalyst was assessed in Diels–Alder cycloaddition reactions and cascade reactions showing excellent catalytic properties.

Mahy, J.-P.

Tetrahedron Lett. 2008, 49, 1865-1869, 10.1016/j.tetlet.2008.01.022

The synthesis of a new cationic iron metalloporphyrin–estradiol conjugate is reported. After a study of its association with the anti-estradiol antibody 7A3 by UV–visible spectroscopy, the influence of the antibody on the sulfoxidation of thioanisole by H2O2 catalyzed by the iron–metalloporphyrin has been investigated.

Dervan, P.B.

Science 1987, 238, 1129-1132, 10.1126/science.3120311

A synthetic 52-residue peptide based on the sequence-specific DNA-binding domain of Hin recombinase (139-190) has been equipped with ethylenediaminetetraacetic acid (EDTA) at the amino terminus. In the presence of Fe(II), this synthetic EDTA-peptide cleaves DNA at Hin recombination sites. The cleavage data reveal that the amino terminus of Hin(139-190) is bound in the minor groove of DNA near the symmetry axis of Hin recombination sites. This work demonstrates the construction of a hybrid peptide combining two functional domains: sequence-specific DNA binding and DNA cleavage.

Kamer, P.C.J.; Laan, W.

Dalton Trans. 2010, 39, 8477, 10.1039/c0dt00239a

The preparation of hybrid transition metalloproteins by thiol-selective incorporation of organometallic rhodium- and ruthenium complexes is described. Phosphine ligands and two rhodium-diphosphine complexes bearing a carboxylic acid group were coupled to the cysteine of PYP R52G, yielding a metalloenzyme active in the rhodium catalyzed hydrogenation of dimethyl itaconate. The successful coupling was shown by 31P NMR spectroscopy and ESI mass spectroscopy. In addition wild-type PYP (PYP WT), PYP R52G and ALBP were successfully modified with a (η6-arene) ruthenium(II) phenanthroline complex via a maleimide linker.

Turner, N.J.; Ward, T.R.

Nat. Chem. 2013, 5, 93-99, 10.1038/NCHEM.1498

Enzymatic catalysis and homogeneous catalysis offer complementary means to address synthetic challenges, both in chemistry and in biology. Despite its attractiveness, the implementation of concurrent cascade reactions that combine an organometallic catalyst with an enzyme has proven challenging because of the mutual inactivation of both catalysts. To address this, we show that incorporation of a d6-piano stool complex within a host protein affords an artificial transfer hydrogenase (ATHase) that is fully compatible with and complementary to natural enzymes, thus enabling efficient concurrent tandem catalysis. To illustrate the generality of the approach, the ATHase was combined with various NADH-, FAD- and haem-dependent enzymes, resulting in orthogonal redox cascades. Up to three enzymes were integrated in the cascade and combined with the ATHase with a view to achieving (i) a double stereoselective amine deracemization, (ii) a horseradish peroxidase-coupled readout of the transfer hydrogenase activity towards its genetic optimization, (iii) the formation of L-pipecolic acid from L-lysine and (iv) regeneration of NADH to promote a monooxygenase-catalysed oxyfunctionalization reaction.

Lu, Y.

J. Am. Chem. Soc. 2014, 136, 11882-11885, 10.1021/ja5054863

Cytochrome c Oxidase (CcO) is known to catalyze the reduction of O2 to H2O efficiently with a much lower overpotential than most other O2 reduction catalysts. However, methods by which the enzyme fine-tunes the reduction potential (E°) of its active site and the corresponding influence on the O2 reduction activity are not well understood. In this work, we report systematic tuning of the heme E° in a functional model of CcO in myoglobin containing three histidines and one tyrosine in the distal pocket of heme. By removing hydrogen-bonding interactions between Ser92 and the proximal His ligand and a heme propionate, and increasing hydrophobicity of the heme pocket through Ser92Ala mutation, we have increased the heme E° from 95 ± 2 to 123 ± 3 mV. Additionally, replacing the native heme b in the CcO mimic with heme a analogs, diacetyl, monoformyl, and diformyl hemes, that posses electron-withdrawing groups, resulted in higher E° values of 175 ± 5, 210 ± 6, and 320 ± 10 mV, respectively. Furthermore, O2 consumption studies on these CcO mimics revealed a strong enhancement in O2 reduction rates with increasing heme E°. Such methods of tuning the heme E° through a combination of secondary sphere mutations and heme substitutions can be applied to tune E° of other heme proteins, allowing for comprehensive investigations of the relationship between E° and enzymatic activity.

Ward, T.R.

Angew. Chem. Int. Ed. 2005, 44, 7764-7767, 10.1002/anie.200502000

The combination of chemical‐ with genetic‐optimization strategies (i.e. chemogenetic) allows the production of artificial hydrogenases based on the biotin–avidin technology. In the spirit of enzymes, second‐coordination‐sphere interactions between the host protein (streptavidin) and the substrate (an olefin) allow fine‐tuning of the selectivity to produce either R or S hydrogenation products.

Anderson, J.L.R.

Curr. Opin. Struct. Biol. 2018, 51, 149-155, 10.1016/j.sbi.2018.04.008

Though established 40 years ago, the field of de novo protein design has recently come of age, with new designs exhibiting an unprecedented level of sophistication in structure and function. With respect to catalysis, de novo enzymes promise to revolutionise the industrial production of useful chemicals and materials, while providing new biomolecules as plug-and-play components in the metabolic pathways of living cells. To this end, there are now de novo metalloenzymes that are assembled in vivo, including the recently reported C45 maquette, which can catalyse a variety of substrate oxidations with efficiencies rivalling those of closely related natural enzymes. Here we explore the successful design of this de novo enzyme, which was designed to minimise the undesirable complexity of natural proteins using a minimalistic bottom-up approach.

Kokubo, T.; Okano, M.

J. Chem. Soc., Chem. Commun. 1983, 0, 769-770, 10.1039/C39830000769

The 1:1 complex between an osmate ester and bovine serum albumin was found to be effective as an enantioselective catalyst in the cis-hydroxylation of alkenes, affording diols in up to 68% e.e. and turnover of the catalyst with t-butyl hydroperoxide.

Mayer, C.; Roelfes, G.

Nat. Rev. Chem. 2019, 3, 687-705, 10.1038/s41570-019-0143-x

The ability of one enzyme to catalyse multiple, mechanistically distinct transformations likely played a crucial role in organisms’ abilities to adapt to changing external stimuli in the past and can still be observed in extant enzymes. Given the importance of catalytic promiscuity in nature, enzyme designers have recently begun to create catalytically promiscuous enzymes in order to expand the canon of transformations catalysed by proteins. This article aims to both critically review different strategies for the design of enzymes that display catalytic promiscuity for new-to-nature reactions and highlight the successes of subsequent directed-evolution efforts to fine-tune these novel reactivities. For the former, we put a particular emphasis on the creation, stabilization and repurposing of reaction intermediates, which are key for unlocking new activities in an existing or designed active site. For the directed evolution of the resulting catalysts, we contrast approaches for enzyme design that make use of components found in nature and those that achieve new reactivities by incorporating synthetic components. Following the critical analysis of selected examples that are now available, we close this Review by providing a set of considerations and design principles for enzyme engineers, which will guide the future generation of efficient artificial enzymes for synthetically useful, abiotic transformations.

Lu, Y.; Zhang, J.-L.

ACS Catal. 2011, 1, 1083-1089, 10.1021/cs200258e

Two questions important to the success in metalloenzyme design are how to attach or anchor metal cofactors inside protein scaffolds and in what way such positioning affects enzymatic properties. We have previously reported a dual anchoring method to position a nonnative cofactor, MnSalen (1), inside the heme cavity of apo sperm whale myoglobin (Mb) and showed that the dual anchoring can increase both the activity and enantioselectivity over single anchoring methods, making this artificial enzyme an ideal system to address the above questions. Here, we report systematic investigations of the effect of different covalent attachment or anchoring positions on reactivity and selectivity of sulfoxidation by the MnSalen-containing Mb enzymes. We have found that changing the left anchor from Y103C to T39C has an almost identical effect of increasing rate by 1.8-fold and increasing selectivity by +15% for S, whether the right anchor is L72C or S108C. At the same time, regardless of the identity of the left anchor, changing the right anchor from S108C to L72C increases the rate by 4-fold and selectivity by +66%. The right anchor site was observed to have a greater influence than the left anchor site on the reactivity and selectivity in sulfoxidation of a wide scope of other ortho-, meta- and para-substituted substrates. The 1·Mb(T39C/L72C) showed the highest reactivity (TON up to 2.32 min–1) and selectivity (ee % up to 83%) among the different anchoring positions examined. Molecular dynamic simulations indicate that these changes in reactivity and selectivity may be due to the steric effects of the linker arms inside the protein cavity. These results indicate that small differences in the anchor positions can result in significant changes in reactivity and enantioselectivity, probably through steric interactions with substrates when they enter the substrate-binding pocket, and that the effects of right and left anchor positions are independent and additive in nature. The finding that the anchoring arms can influence both the positioning of the cofactor and steric control of substrate entrance will help design better functional metalloenzymes with predicted catalytic activity and selectivity.

Happe, T.; Hemschemeier, A.

Nat. Rev. Chem. 2018, 2, 231-243, 10.1038/s41570-018-0029-3

Metal cofactors considerably widen the catalytic space of naturally occurring enzymes whose specific and enantioselective catalytic activity constitutes a blueprint for economically relevant chemical syntheses. To optimize natural enzymes and uncover novel reactivity, we need a detailed understanding of cofactor–protein interactions, which can be challenging to obtain in the case of enzymes with sophisticated cofactors. As a case study, we summarize recent research on the [FeFe]-hydrogenases, which interconvert protons, electrons and dihydrogen at a unique iron-based active site. We can now chemically synthesize the complex cofactor and incorporate it into an apo-protein to afford functional enzymes. By varying both the cofactor and the polypeptide components, we have obtained detailed knowledge on what is required for a metal cluster to process H2. In parallel, the design of artificial proteins and catalytically active nucleic acids are advancing rapidly. In this Perspective, we introduce these fields and outline how chemists and biologists can use this knowledge to develop novel tailored semisynthetic catalysts.

Cavazza, C.; Ménage, S.

ChemBioChem 2009, 10, 545-552, 10.1002/cbic.200800595

Magic Mn–salen metallozyme: The design of an original, artificial, inorganic, complex‐protein adduct, has led to a better understanding of the synergistic effects of both partners. The exclusive formation of sulfoxides by the hybrid biocatalyst, as opposed to sulfone in the case of the free inorganic complex, highlights the modulating role of the inorganic‐complex‐binding site in the protein.

Sheldon, R.A.

Biotechnol. Bioeng. 2000, 67, 87-96, 10.1002/(SICI)1097-0290(20000105)67:1<87::AID-BIT10>3.0.CO;2-8

A semisynthetic peroxidase was designed by exploiting the structural similarity of the active sites of vanadium dependent haloperoxidases and acid phosphatases. Incorporation of vanadate ion into the active site of phytase (E.C. 3.1.3.8), which mediates in vivo the hydrolysis of phosphate esters, leads to the formation of a semisynthetic peroxidase, which catalyzes the enantioselective oxidation of prochiral sulfides with H2O2 affording the S‐sulfoxide, e.g. in 66% ee at 100% conversion for thioanisole. Under reaction conditions the semi‐synthetic vanadium peroxidase is stable for over 3 days with only a slight decrease in turnover frequency. Polar water‐miscible cosolvents, such as methanol, dioxane, and dimethoxyethane, can be used in concentrations of 30% (v/v) at a small penalty in activity and enantioselectivity. Among the transition metal oxoanions that are known to be potent inhibitors, only vanadate resulted in a semisynthetic peroxidase when incorporated into phytase. A number of other acid phosphatases and hydrolases were tested for peroxidase activity, when incorporated with vanadate ion. Phytases from Aspergillus ficuum, A. fumigatus, and A. nidulans, sulfatase from Helix pomatia, and phospholipase D from cabbage catalyzed enantioselective oxygen transfer reactions when incorporated with vanadium. However, phytase from A. ficuum was unique in also catalyzing the enantioselective sulfoxidation, albeit at a lower rate, in the absence of vanadate ion.

Imanaka, T.

FEBS Lett. 1995, 375, 273-276, 10.1016/0014-5793(95)01224-3

In order to engineer a new type of catalytic antibody, we attempt to use a monoclonal antibody L chain as a host protein for a porphyrin. TCPP (meso‐tetrakis(4‐carboxyphenyl)porphyine) was chemically synthesized and Balb/c mice were immunized using TCPP as a hapten. Two hybridoma cells (03‐1, 13‐1), that produce monoclonal antibody against TCPP, were obtained. Genes for both H and L chains of monoclonal antibodies were cloned, sequenced and overexpressed using E. coli as a host. ELISA and fluorescence quenching method show that the independent antibody L chains from both Mab03‐1 and Mab13‐1 have specific interaction with TCPP. Furthermore, the recombinant antibody L chain from Mab13‐1 exhibits much higher peroxidase activity than TCPP Fe(III) alone. The enzyme activity was detectable with pyrogallol and ABTS (2,2‐azinobis‐3‐ethylbenzthiazolin‐6‐sulfonic acid) but not with catechol. This new catalytic antibody was extremely thermostable. Optimum temperature of the peroxidase reaction by the complex of 13‐1L chain and TCPP Fe(III) was 90°C, while that the TCPP Fe(III) alone was 60°C.

Lin, Y.-W.

ACS Catal. 2019, 9, 7888-7893, 10.1021/acscatal.9b02226

Approaches to degradation of industrial dyes are desirable, of which bioremediation is more favorable. In addition to the use of native enzymes, rational design of artificial enzymes provides an alternative approach. Meanwhile, few designs can achieve a catalytic activity comparable to that of native enzymes. We have previously designed two generations of artificial dye-decolorizing peroxidases (DyPs) in myoglobin (Mb) by introduction of Tyr43 and Trp138 in the heme pocket; however, the activity is moderate. To improve the activity of the artificial DyP, we herein designed a third generation by introduction of an additional Trp (P88W) to the protein surface, named F43Y/F138W/P88W Mb. The third generation of artificial DyP was shown to exhibit a catalytic efficiency exceeding that of various native DyPs and comparable to that of the most efficient native DyPs. Titration of reactive blue 19 (RB19) and molecular docking studies revealed crucial roles of Trp88 in substrate binding and oxidation, which acts as a catalytic site. This study not only provides clues for heme protein design but also suggests that the artificial DyP has potential applications for bioremediation in the future.

Keinan, E.

Pure Appl. Chem. 1990, 62, 2013-2019, 10.1351/pac199062102013

An attempt was made to mimic cytochrome P-450-like activity using antibodies elicited against metallo-porphyrins. Monoclonal antibodies raised against a water-soluble Sn(1V) porphyrin complex (1) exhibited Specificity for a variety of monomeric metalloporphyrins, as well as for the b-0x0-Fe(III) porphyrin dimer 2. Some antibodies were found to be more selective for the monomer 1 than for the dimer 2, suggesting an "edge-on" recognition of the planar porphyrin molecule. The catalytic activity of the antibody-metalloporphyrin complexes was investigated using the epoxidation of styrene by iodosobenzene as a model reaction. Three biphasic media were studied for this reaction: reverse micelles, microemulsions, and solid catalyst in organic solvent. The most promising results were obtained with solid catalyst (obtained via lyophilization of equimolar amounts of Mn(TCP)Cl and specific antibody) in dry CHzClz at room temperature, as indicated by the high turnover numbers of the catalyst. A difference in the relative activity of the various monoclonal antibodies (MABs) was noted. The anti-1 antibodies displayed ca. 30-60% higher activity compared to a nonrelevant MAB.

Schwaneberg, U.

Angew. Chem. Int. Ed. 2019, 58, 4454-4464, 10.1002/anie.201811042

Incorporating artificial metal‐cofactors into protein scaffolds results in a new class of catalysts, termed biohybrid catalysts or artificial metalloenzymes. Biohybrid catalysts can be modified chemically at the first coordination sphere of the metal complex, as well as at the second coordination sphere provided by the protein scaffold. Protein‐scaffold reengineering by directed evolution exploits the full power of nature's diversity, but requires validated screening and sophisticated metal cofactor conjugation to evolve biohybrid catalysts. In this Minireview, we summarize the recent efforts in this field to establish high‐throughput screening methods for biohybrid catalysts and we show how non‐chiral catalysts catalyze reactions enantioselectively by highlighting the first successes in this emerging field. Furthermore, we shed light on the potential of this field and challenges that need to be overcome to advance from biohybrid catalysts to true artificial metalloenzymes.