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.

Brustad, E.M.; Nicewicz, D.A.

ChemBioChem 2020, 21, 3146-3150, 10.1002/cbic.202000362

A pair of 9-mesityl-10-phenyl acridinium (Mes−Acr+) photoredox catalysts were synthesized with an iodoacetamide handle for cysteine bioconjugation. Covalently tethering of the synthetic Mes−Acr+ cofactors with a small panel of thermostable protein scaffolds resulted in 12 new artificial enzymes. The unique chemical and structural environment of the protein hosts had a measurable effect on the photophysical properties and photocatalytic activity of the cofactors. The constructed Mes−Acr+ hybrid enzymes were found to be active photoinduced electron-transfer catalysts, controllably oxidizing a variety of aryl sulfides when irradiated with visible light, and possessed activities that correlated with the photophysical characterization data. Their catalytic performance was found to depend on multiple factors including the Mes−Acr+ cofactor, the protein scaffold, the location of cofactor immobilization, and the substrate. This work provides a framework toward adapting synthetic photoredox catalysts into artificial cofactors and includes important considerations for future bioengineering efforts.

Creus, M.; Ward, T.R.

Prog. Inorg. Chem. 2011, 203-253, 10.1002/9781118148235.ch4

n/a

Sheldon, R.A.

Chem. Commun. 1998, 1891-1892, 10.1039/a804702b

Phytase (E.C. 3.1.3.8), which in vivo mediates the hydrolysis of phosphate esters, catalyses the enantioselective oxidation of thioanisole with H2O2, both in the presence and absence of vanadate ion, affording the S-sulfoxide in up to 66% ee at 100% conversion.

Sheldon, R.A.

Can. J. Chem. 2002, 80, 622-625, 10.1139/v02-082

The catalytic Zn2+ ion was extracted from thermolysin, which had been covalently bound to Eupergit C. The apo-enzyme incorporated the oxometallate anions MoO42–, SeO42–, and WO42– with partial restoration of the proteolytic activity. Tungstate thermolysin was moderately active in the sulfoxidation of thioanisole by hydrogen peroxide, whereas its activity towards phenylmercaptoacetophenone, which was designed to bind well in the active site of thermolysin, was much higher.

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.

Sheldon, R.A.

Top. Catal. 2000, 13, 259-265, 10.1023/A:1009094619249

Approaches to the rational design of vanadium-based biocatalytic and biomimetic model systems as catalysts for enantioselective oxidations are reviewed. Incorporation of vanadate ion into the active site of phytase (E.C. 3.1.3.8), which in vivo mediates the hydrolysis of phosphate esters, afforded a relatively stable and inexpensive semi-synthetic peroxidase. It catalysed the enantioselective oxidation of prochiral sulfides with H2O2 affording the S-sulfoxide, e.g., in 68% ee at 100% conversion for thioanisole. Amongst the transition metal oxoanions that are known to be potent inhibitors of phosphatases, only vanadate resulted in a semi-synthetic peroxidase, when incorporated into phytase. In a biomimetic approach, vanadium complexes of chiral Schiff's base complexes were encapsulated in the super cages of a hydrophobic zeolite Y. Unfortunately, these ship-in-a-bottle complexes afforded only racemic sulfoxide in the catalytic oxidation of thioanisole with H2O2.