Mahy, J.-P.

Biotechnol. Appl. Biochem. 2020, 67, 563-573, 10.1002/bab.1906

We report a new artificial hydrogenase made by covalent anchoring of the iron Knölker's complex to a xylanase S212C variant. This artificial metalloenzyme was found to be able to catalyze efficiently the transfer hydrogenation of the benchmark substrate trifluoroacetophenone by sodium formate in water, yielding the corresponding secondary alcohol as a racemic. The reaction proceeded more than threefold faster with the XlnS212CK biohybrid than with the Knölker's complex alone. In addition, efficient conversion of trifluoroacetophenone to its corresponding alcohol was reached within 60 H with XlnS212CK, whereas a ≈2.5-fold lower conversion was observed with Knölker's complex alone as catalyst. Moreover, the data were rationalized with a computational strategy suggesting the key factors of the selectivity. These results suggested that the Knölker's complex was most likely flexible and could experience free rotational reorientation within the active-site pocket of Xln A, allowing it to access the subsite pocket populated by trifluoroacetophenone.

DeGrado, W.F.; Lombardi, A.

C. R. Chim. 2007, 10, 703-720, 10.1016/j.crci.2007.03.010

A major objective in protein science is the design of enzymes with novel catalytic activities that are tailored to specific applications. Such enzymes may have great potential in biocatalysis and biosensor technology, such as in degradation of pollutants and biomass, and in drug and food processing. To reach this objective, investigations into the basic biochemical functioning of metalloproteins are still required. In this perspective, metalloprotein design provides a powerful approach first to contribute to a more comprehensive understanding of the way metalloproteins function in biology, with the ultimate goal of developing novel biocatalysts and sensing devices. Metalloprotein mimetics have been developed through the introduction of novel metal-binding sites into naturally occurring proteins as well as through de novo protein design. We have approached the challenge of reproducing metalloprotein active sites by using a miniaturization process. We centered our attention on iron-containing proteins, and we developed models for heme proteins and diiron–oxo proteins. In this paper we summarize the results we obtained on the design, structural, and functional properties of DFs, a family of artificial diiron proteins.

Fasan, R.

Curr. Opin. Green Sustain. Chem. 2021, 31, 100494, 10.1016/j.cogsc.2021.100494

The direct functionalization of C–H bonds constitutes a powerful strategy to construct and diversify organic molecules. However, controlling the chemo- and site-selectivity of this transformation, particularly in complex molecular settings, represents a significant challenge. Metalloenzymes are ideal platforms for achieving catalyst-controlled selective C–H bond functionalization as their reactivities can be tuned by protein engineering and/or redesign of their cofactor environment. In this review, we highlight recent progress in the development of engineered and artificial metalloenzymes for C–H functionalization, with a focus on biocatalytic strategies for selective C–H oxyfunctionalization and halogenation as well as C–H amination and C–H carbene insertion via abiological nitrene and carbene transfer chemistries. Engineered heme and nonheme iron dependent enzymes have emerged as promising scaffolds for executing these transformations with high chemo-, regio-, and stereocontrol as well as tunable selectivity. These emerging systems and methodologies have expanded the toolbox of sustainable strategies for organic synthesis and created new opportunities for the generation of chiral building blocks, the late-stage C–H functionalization of complex molecules, and the total synthesis of natural products.

Yilmaz, F.

Process Biochem. 2018, 72, 71-78, 10.1016/j.procbio.2018.06.005

Carbonic anhydrase (carbonic dehydratase) (CA) is a metalloenzyme that contains zinc (Zn2+) ion in its active site. CA catalyzes the reversible conversion of carbon dioxide and water to bicarbonate and protons. Zn2+ ions, which are present in the active site of the enzyme, interact with the substrate molecules directly and cause catalytic effect. In this study, a nano-enzyme system was designed in aqueous solutions at room temperature and under nitrogen atmosphere to use the CA enzyme without any pre-treatment and deformation in its structure. The novel concept ANADOLUCA (AmiNo Acid (monomer) Decorated and Light Underpinning Conjugation Approach) was used for this process, nano CA enzyme of size 93 nm was synthesized. The activity of the synthesized nano CA was measured following the change in absorbance during the conversion of 4-nitrophenylacetate (NPA) to 4-nitrophenylate ion at 348 nm for a period of 10 min at 25 °C compared with free CA enzyme. Km and Vmax values for nano CA enzyme were found to be 0.442 mM and 1.6 × 10−3 mM min-1, respectively, whereas Km and Vmax values for free CA were found to be 0.471 mM and 1.5 × 10−3 mM min-1, respectively. In addition to these, the Zn2+ ion present in the active site of the nano CA enzyme was replaced by rodium metal. This nanorodium-substituted CA has been investigated as a new reductase enzyme for the stereoselective hydrogenation of olefins. Then, the Zn2+ ion in the active site of the nano CA enzyme was replaced with manganese metal to enhance the enzyme structure, thereby gaining characteristics of peroxidase. This newly synthesized nano manganese-substituted CA enzyme was investigated for its role as a peroxidase, which could be an alternative for hydrogen peroxidases.

Mahy, J.-P.

C. R. Chim. 2007, 10, 684-702, 10.1016/j.crci.2006.12.014

Catalytic antibodies with a metalloporphyrin cofactor or “hemoabzymes”, used as models for hemoproteins like peroxidases and cytochrome P450s, represent a promising route to catalysts tailored for selective oxidation reactions. The first strategy has been to produce anti-porphyrin antibodies, raised against various N-substituted- and meso-carboxyaryl-porphyrins, which led to monoclonal antibodies exhibiting, in the presence of the corresponding iron-porphyrin cofactor, a significant peroxidase activity. We ourselves obtained an artificial hemoprotein by associating a monoclonal antibody, 13G10, and its iron(III)-α,α,α,β-meso-tetrakis(ortho-carboxyphenyl)porphyrin (Fe(ToCPP)) hapten, which exhibited a significant peroxidase activity. Biological studies suggested that in this antibody, a carboxylic acid side chain of the protein participated in the catalysis, but no amino acid residue acting as an axial ligand of the iron was detected. Therefore, to provide the iron atom with an axial ligand, we raised antibodies against microperoxidase 8, a heme octapeptide containing a histidine bound to the iron atom. This strategy was successful, as an antibody–microperoxidase 8 complex (3A3–MP8) led to the best kcat/Km ever reported for antibody–porphyrin complexes. The ability of the 3A3–MP8 complex to catalyze the selective oxidation of substrates was studied and it was found able to catalyze the regioselective nitration of aromatics by NO2−/H2O2 as well as the stereoselective oxidation of sulfides like thioanisole by H2O2. Other strategies based on antibodies have to be developed to obtain more efficient biomimetic systems for cytochrome P450s. A first one could involve the modification of anti-substrate antibodies by covalent linkage of an iron(III)-porphyrin close to the binding site of the substrate, to obtain an artificial hemoprotein able to catalyze its regioselective oxidation.

Lin, Y.-W.

Biotechnol. Appl. Biochem. 2019, 10.1002/bab.1788

Environmental pollutants, such as industrial dyes and halophenols, are harmful to human health, which urgently demand degradation. Bioremediation has been shown to be a cost‐effective and ecofriendly approach. As reviewed herein, significant progress has been made in the last decade for biodegradation of both industrial dyes and halophenols, by engineering of native dye‐decolorizing peroxidases (DyPs) and dehaloperoxidases (DHPs), and by design of artificial heme enzymes in both native and de novo protein scaffolds. The catalytic efficiency of artificial DyPs and DHPs can be rationally designed comparable to or even beyond those of natural counterparts. The enzymes are on their way from laboratory to industry and will play more crucial roles in environmental protection toward a green future.

Ward, T.R.

C. R. Chim. 2007, 10, 678-683, 10.1016/j.crci.2007.02.020

We report on our efforts to create efficient artificial metalloenzymes for the enantioselective hydrogenation of N-protected dehydroamino acids using streptavidin as host protein. Introduction of an (R)-proline spacer between the biotin anchor and the diphosphine moiety affords a versatile ligand Biot-(R)-Pro-1 which displays good (S)-selectivities in the presence of streptavidin (91% ee). The resulting artificial metalloenzyme [Rh(Biot-(R)-Pro-1)(COD)]+ ⊂ WT-Sav displays increased stability against organic solvents.

Berggren, G.

Curr. Opin. Green Sustain. Chem. 2021, 32, 100521, 10.1016/j.cogsc.2021.100521

Hydrogenases are gas processing redox enzymes central in hydrogen metabolism. The interdisciplinary nature of hydrogenase research is underscored by the development of “artificial maturation”, enabling the preparation of semi-synthetic hydrogenases through the incorporation of synthetic cofactors into a range of apo-hydrogenase hosts under in vitro and in vivo conditions. Herein, we discuss how the preparation of such semi-synthetic [FeFe]-hydrogenases has elucidated structural elements of the cofactor critical for catalysis and reactivity towards known inhibitors. It has also provided a convenient method for exploring the biodiversity of this enzyme family and thereby facilitated investigation of the role of the outer-coordination sphere in tuning the reactivity of the H-cluster. In parallel, hijacking the assembly line of the [FeFe]-hydrogenase through incorporation of synthetic precursors has provided detailed insight into the biosynthesis of the H-cluster. Moreover, it has allowed the preparation of Mn analogs of [Fe] hydrogenase.

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.