DeGrado, W.F.; Lombardi, A.

Eur. J. Inorg. Chem. 2015, 2015, 3371-3390, 10.1002/ejic.201500470

A single polypeptide chain may provide an astronomical number of conformers. Nature selected only a trivial number of them through evolution, composing an alphabet of scaffolds, that can afford the complete set of chemical reactions needed to support life. These structural templates are so stable that they allow several mutations without disruption of the global folding, even having the ability to bind several exogenous cofactors. With this perspective, metal cofactors play a crucial role in the regulation and catalysis of several processes. Nature is able to modulate the chemistry of metals, adopting only a few ligands and slightly different geometries. Several scaffolds and metal‐binding motifs are representing the focus of intense interest in the literature. This review discusses the widespread four‐helix bundle fold, adopted as a scaffold for metal binding sites in the context of de novo protein design to obtain basic biochemical components for biosensing or catalysis. In particular, we describe the rational refinement of structure/function in diiron–oxo protein models from the due ferri (DF) family. The DF proteins were developed by us through an iterative process of design and rigorous characterization, which has allowed a shift from structural to functional models. The examples reported herein demonstrate the importance of the synergic application of de novo design methods as well as spectroscopic and structural characterization to optimize the catalytic performance of artificial enzymes.

Creus, M.

Protein Expression Purif. 2011, 77, 131-139, 10.1016/j.pep.2011.01.003

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.

Jaussi, R.

Protein Expression Purif. 2014, 93, 54-62, 10.1016/j.pep.2013.10.015

Artificial metalloenzymes result from the incorporation of a catalytically competent biotinylated organometallic moiety into full-length (i.e. mature) streptavidin. With large-scale industrial biotechnology applications in mind, large quantities of recombinant streptavidin are required. Herein we report our efforts to produce wild-type mature and biotin-free streptavidin using the yeast Pichia pastoris expression system. The streptavidin gene was inserted into the expression vector pPICZαA in frame with the Saccharomyces cerevisiae α-mating factor secretion signal. In a fed-batch fermentation using a minimal medium supplemented with trace amounts of biotin, functional streptavidin was secreted at approximately 650 mg/L of culture supernatant. This yield is approximately threefold higher than that from Escherichia coli, and although the overall expression process takes longer (ten days vs. two days), the downstream processing is simplified by eliminating denaturing/refolding steps. The purified streptavidin bound ∼3.2 molecules of biotin per tetramer. Upon incorporation of a biotinylated piano-stool catalyst, the secreted streptavidin displayed identical properties to streptavidin produced in E. coli by showing activity as artificial imine reductase.

Ward, T.R.

Eur. J. Inorg. Chem. 2015, 2015, 3406-3418, 10.1002/ejic.201500408

In the past decade, artificial metalloenzymes (AMEs) have emerged as attractive alternatives to more traditional homogeneous catalysts and enzymes. This microreview presents a selection of recent achievements in the design of such hybrid catalysts. These include artificial zinc hydrolases and metathesases, the heme‐protein repurposing for C–H, N–H, and S–H insertion reactions, novel light‐driven redox hybrid catalysts, novel scaffold proteins, and metallocofactor anchoring techniques and metalloenzyme models.

Salmain, M.

Eur. J. Inorg. Chem. 2018, 2018, 1383-1393, 10.1002/ejic.201701359

Biohybrid catalysts resulting from the dative anchoring of half‐sandwich organometallic complexes [M(arene)(H2O)x(Cl)y]n+ (M = RuII, arene = η6‐benzene, p‐cymene or mesitylene; M = IrIII, RhIII, arene = η5‐Cp*; x = 1–3, y = 0–2, n = 0–2) to bovine beta‐lactoglobulin (βLG) or hen egg white lysozyme showed unprecedented behaviour. These constructs were shown to catalyse the asymmetric transfer hydrogenation of aryl ketones in water with sodium formate as hydrogen donor at a much faster rate than the complexes alone. Full conversion of the benchmark substrate 2,2,2‐trifluoroacetophenone was reached with an ee of 86 % for the most selective biohybrid. Surprisingly, even the crude biohybrid gave a good ee despite the presence of non‐protein‐bound metal species in the reaction medium. Other aryl ketones were reduced in the same way, and the highest ee was obtained for ortho‐substituted acetophenone derivatives. Furthermore, treatment of βLG with dimethyl pyrocarbonate resulted in a noticeable decrease of the activity and selectivity of the biohybrid, indicating that the sole accessible histidine residue (His146) was probably involved in the coordination and activation of Ru(benzene). This work underscores that protein scaffolds are efficient chiral ligands for asymmetric catalysis. The use of sodium formate instead of dihydrogen makes this approach safe, inexpensive and environmentally friendly.

Fontecilla-Camps, J.C.

Eur. J. Inorg. Chem. 2013, 2013, 3596-3600, 10.1002/ejic.201300592

The crystal structure of bovine β‐lactoglobulin bound to a complex consisting of a (η5‐Cp*)Rh(2,2′‐dipyridylamine) head and a lauric acid derived hydrophobic tail has been solved at 1.85 Å resolution. Previous work has shown that this hybrid catalyzes the transfer hydrogenation of an aryl ketone in neat water with formate as hydrogen donor with enantiomeric excess (ee) of about 26 %. Calculations using the X‐ray model indicate that the complex head can adopt discrete conformations, which may explain the ee observed.

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.