Artificial Gold Enzymes Using a Genetically Encoded Thiophenol-Based Noble-Metal-Binding Ligand.

Incorporating noble metals in artificial metalloenzymes (ArMs) is challenging due to the lack of suitable soft coordinating ligands among natural amino acids. We present a new class of ArMs featuring a genetically encoded noble-metal-binding site based on a non-canonical thiophenol-based amino acid, 4-mercaptophenylalanine (pSHF), incorporated in the transcriptional regulator LmrR through stop codon suppression. We demonstrate that pSHF is an excellent ligand for noble metals in their low oxidation states.

FhuA: From Iron-Transporting Transmembrane Protein to Versatile Scaffolds through Protein Engineering.

Protein engineering has emerged as a powerful methodology to tailor the properties of proteins. It empowers the design of biohybrid catalysts and materials, thereby enabling the convergence of materials science, chemistry, and medicine. The choice of a protein scaffold is an important factor for performance and potential applications.

Protein Nanopore Membranes Prepared by a Simple Langmuir-Schaefer Approach.

Filtration through membranes with nanopores is typically associated with high transmembrane pressures and high energy consumption. This problem can be addressed by reducing the respective membrane thickness. Here, a simple procedure is described to prepare ultrathin membranes based on protein nanopores, which exhibit excellent water permeance, two orders of magnitude superior to comparable, industrially applied membranes.

Fe(III)-complex mediated bacterial cell surface immobilization of eGFP and enzymes.

We report a facile and reversible method to immobilize a broad range of His6-tagged proteins on the E. coli cell surface through Fe(iii)-metal complexes. A His6-tagged eGFP and four His6-tagged enzymes were successfully immobilized on the cell surface.

An artificial ruthenium-containing β-barrel protein for alkene-alkyne coupling reaction.

A modified Cp*Ru complex, equipped with a maleimide group, was covalently attached to a cysteine of an engineered variant of Ferric hydroxamate uptake protein component: A (FhuA). This synthetic metalloprotein catalyzed the intermolecular alkene-alkyne coupling of 3-butenol with 5-hexynenitrile. When compared with the protein-free Cp*Ru catalyst, the biohybrid catalyst produced the linear product with higher regioselectivity.

A Photoclick-Based High-Throughput Screening for the Directed Evolution of Decarboxylase OleT.

Enzymatic oxidative decarboxylation is an up-and-coming reaction yet lacking efficient screening methods for the directed evolution of decarboxylases. Here, we describe a simple photoclick assay for the detection of decarboxylation products and its application in a proof-of-principle directed evolution study on the decarboxylase OleT. The assay was compatible with two frequently used OleT operation modes (directly using hydrogen peroxide as the enzyme's co-substrate or using a reductase partner) and the screening of saturation mutagenesis libraries identified two enzyme variants shifting the enzyme's substrate preference from long chain fatty acids toward styrene derivatives.

Engineering of Laccase CueO for Improved Electron Transfer in Bioelectrocatalysis by Semi-Rational Design.

Invited for the cover of this issue is the group of Ulrich Schwaneberg at the Institute of Biotechnology, RWTH-Aachen University and DWI Lebniz Institute of Interactive Materials. The picture calls for special attention to be paid to the extra Cu binding site of Copper efflux Oxidase (CueO), due to its predominant function in tuning the electrocatalytic kinetics towards oxygen reduction. Read the full text of the article at 10.

Engineering of Laccase CueO for Improved Electron Transfer in Bioelectrocatalysis by Semi-Rational Design.

Copper efflux oxidase (CueO) from Escherichia coli is a special bacterial laccase due to its fifth copper binding site. Herein, it is discovered that the fifth Cu occupancy plays a crucial and favorable role of electron relay in bioelectrocatalytic oxygen reduction. By substituting the residues at the four coordinated positions of the fifth Cu, 11 beneficial variants are identified with ≥2.

Directed aryl sulfotransferase evolution toward improved sulfation stoichiometry on the example of catechols.

Sulfation is an important way for detoxifying xenobiotics and endobiotics including catechols. Enzymatic sulfation occurs usually with high chemo- and/or regioselectivity under mild reaction conditions. In this study, a two-step p-NPS-4-AAP screening system for laboratory evolution of aryl sulfotransferase B (ASTB) was developed in 96-well microtiter plates to improve the sulfate transfer efficiency toward catechols.

Anchor Peptide-Mediated Surface Immobilization of a Grubbs-Hoveyda-Type Catalyst for Ring-Opening Metathesis Polymerization.

Adhesion promoting peptides have been reported to enable efficient enzyme immobilization on various material surfaces. Here we report the first immobilization of a synthetic Grubbs-Hoveyda (GH) type catalyst on two different materials (silica and polypropylene). To this end, the GH catalyst was coupled to an engineered (F16C) variant of the adhesion promoting peptide LCI through thiol-maleimide "click" reaction.

Olefin metathesis catalysts embedded in β-barrel proteins: creating artificial metalloproteins for olefin metathesis.

This review summarizes the recent progress of Grubbs-Hoveyda (GH) type olefin metathesis catalysts incorporated into the robust fold of β-barrel proteins. Anchoring strategies are discussed and challenges and opportunities in this emerging field are shown from simple small-molecule transformations over ring-opening metathesis polymerizations to in vivo olefin metathesis.

Towards the Evolution of Artificial Metalloenzymes-A Protein Engineer's Perspective.

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.

A hydroquinone-specific screening system for directed P450 evolution.

The direct hydroxylation of benzene to hydroquinone (HQ) under mild reaction conditions is a challenging task for chemical catalysts. Cytochrome P450 (CYP) monooxygenases are known to catalyze the oxidation of a variety of aromatic compounds with atmospheric dioxygen. Protein engineering campaigns led to the identification of novel P450 variants, which yielded improvements in respect to activity, specificity, and stability.

Directed OmniChange Evolution Converts P450 BM3 into an Alkyltrimethylammonium Hydroxylase.

Cetyl-trimethylammonium bromide (CTAB) is a widely used cationic surfactant that is biodegradable in nature. CTAB biodegradation requires hydroxylation in the first step, which is rate-limiting and crucial for solubility in water. In this study, the OmniChange multi-site mutagenesis method was applied to reengineer the P450 BM3 substrate specificity towards the hydroxylation of CTAB by simultaneous mutagenesis of four previously reported positions (R47, Y51, F87, and L188).

2-Methyl-2,4-pentanediol (MPD) boosts as detergent-substitute the performance of ß-barrel hybrid catalyst for phenylacetylene polymerization.

Covering hydrophobic regions with stabilization agents to solubilize purified transmembrane proteins is crucial for their application in aqueous media. The small molecule 2-methyl-2,4-pentanediol (MPD) was used to stabilize the transmembrane protein (FhuA) utilized as host for the construction of a rhodium-based biohybrid catalyst. Unlike commonly used detergents such as sodium dodecyl sulfate or polyethylene polyethyleneglycol, MPD does not form micelles in solution.

Artificial Diels-Alderase based on the transmembrane protein FhuA.

Copper(I) and copper(II) complexes were covalently linked to an engineered variant of the transmembrane protein Ferric hydroxamate uptake protein component A (FhuA ΔCVF(tev)). Copper(I) was incorporated using an N-heterocyclic carbene (NHC) ligand equipped with a maleimide group on the side arm at the imidazole nitrogen. Copper(II) was attached by coordination to a terpyridyl ligand.

Selective Metal-Free Hydrosilylation of CO2 Catalyzed by Triphenylborane in Highly Polar, Aprotic Solvents.

Triphenylborane (BPh3 ) in highly polar, aprotic solvents catalyzes hydrosilylation of CO2 effectively under mild conditions to provide silyl formates with high chemoselectivity (>95 %) and without over-reduction. This system also promotes reductive hydrosilylation of tertiary amides as well as dehydrogenative coupling of silane with alcohols.

Construction of a hybrid biocatalyst containing a covalently-linked terpyridine metal complex within a cavity of aponitrobindin.

A hybrid biocatalyst containing a metal terpyridine (tpy) complex within a rigid β-barrel protein nitrobindin (NB) is constructed. A tpy ligand with a maleimide group, N-[2-([2,2':6',2''-terpyridin]-4'-yloxy)ethyl]maleimide (1), was covalently linked to Cys96 inside the cavity of NB to prepare a conjugate NB-1. Binding of Cu(2+), Zn(2+), or Co(2+) ion to the tpy ligand in NB-1 was confirmed by UV-vis spectroscopy and ESI-TOF MS measurements.

Hybrid ruthenium ROMP catalysts based on an engineered variant of β-barrel protein FhuA ΔCVF(tev) : effect of spacer length.

A biohybrid ring-opening olefin metathesis polymerization catalyst based on the reengineered β-barrel protein FhuA ΔCVF(tev) was chemically modified with respect to the covalently anchored Grubbs-Hoveyda type catalyst. Shortening of the spacer (1,3-propanediyl to methylene) between the N-heterocyclic carbene ligand and the cysteine site 545 increased the ROMP activity toward a water-soluble 7-oxanorbornene derivative. The cis/trans ratio of the double bond in the polymer was influenced by the hybrid catalyst.