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.

Approaching boiling point stability of an alcohol dehydrogenase through computationally-guided enzyme engineering.

Enzyme instability is an important limitation for the investigation and application of enzymes. Therefore, methods to rapidly and effectively improve enzyme stability are highly appealing. In this study we applied a computational method (FRESCO) to guide the engineering of an alcohol dehydrogenase.

A closer look into NADPH oxidase inhibitors: Validation and insight into their mechanism of action.

NADPH-oxidases (NOXs) purposefully produce reactive-oxygen-species (ROS) and are found in most kingdoms of life. The seven human NOXs are each characterized by a specific expression profile and a fine regulation to spatio-temporally tune ROS concentration in cells and tissues. One of the best known roles for NOXs is in host protection against pathogens but ROS themselves are important second messengers involved in tissue regeneration and the modulation of pathways that induce and sustain cell proliferation.

Convergent Cascade Catalyzed by Monooxygenase-Alcohol Dehydrogenase Fusion Applied in Organic Media.

With the aim of applying redox-neutral cascade reactions in organic media, fusions of a type II flavin-containing monooxygenase (FMO-E) and horse liver alcohol dehydrogenase (HLADH) were designed. The enzyme orientation and expression vector were found to influence the overall fusion enzyme activity. The resulting bifunctional enzyme retained the catalytic properties of both individual enzymes.

Design of Artificial Alcohol Oxidases: Alcohol Dehydrogenase-NADPH Oxidase Fusions for Continuous Oxidations.

To expand the arsenal of industrially applicable oxidative enzymes, fusions of alcohol dehydrogenases with an NADPH-oxidase were designed. Three different alcohol dehydrogenases (LbADH, TbADH, ADHA) were expressed with a thermostable NADPH-oxidase fusion partner (PAMO C65D) and purified. The resulting bifunctional biocatalysts retained the catalytic properties of the individual enzymes, and acted essentially like alcohol oxidases: transforming alcohols to ketones by using dioxygen as mild oxidant, while merely requiring a catalytic amount of NADP .

Enzyme Fusions in Biocatalysis: Coupling Reactions by Pairing Enzymes.

One approach to bringing enzymes together for multienzyme biocatalysis is genetic fusion. This enables the production of multifunctional enzymes that can be used for whole-cell biotransformations or for in vitro (cascade) reactions. In some cases and in some aspects, such as expression and conversions, the fused enzymes outperform a combination of the individual enzymes.

Coupled reactions by coupled enzymes: alcohol to lactone cascade with alcohol dehydrogenase-cyclohexanone monooxygenase fusions.

The combination of redox enzymes for redox-neutral cascade reactions has received increasing appreciation. An example is the combination of an alcohol dehydrogenase (ADH) with a cyclohexanone monooxygenase (CHMO). The ADH can use NADP to oxidize cyclohexanol to form cyclohexanone and NADPH.