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. In contrast, some enzyme fusions are greatly compromised in activity and/or expression. In this Minireview, we give an overview of studies on fusions between two or more enzymes that were used for biocatalytic applications, with a focus on oxidative enzymes. Typically, the enzymes are paired to facilitate cofactor recycling or cosubstrate supply. In addition, different linker designs are briefly discussed. Although enzyme fusion is a promising tool for some biocatalytic applications, future studies could benefit from integrating the findings of previous studies in order to improve reliability and effectiveness.

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+ . In small-scale reactions, the purified fusion enzymes show good performances, with 69-99 % conversion, 99 % ee with a racemic substrate, and high cofactor and enzyme total turnover numbers. As the fusion enzymes essentially act as oxidases, we found that commonly used high-throughput oxidase-activity screening methods can be used. Therefore, if needed, the fusion enzymes could be easily engineered to tune their properties.

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. The lyophilized cell-free extract containing the bifunctional enzyme was applied for the convergent cascade reaction consisting of cyclobutanone and butane-1,4-diol in different microaqueous media with only 5 % (v/v) aqueous buffer without any addition of external cofactor. Methyl tert-butyl ether and cyclopentyl methyl ether were found to be the best organic media for the synthesis of γ-butyrolactone, resulting in about 27 % analytical yield.

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. Both products are then used by CHMO to produce ε-caprolactone. In this study, these two redox-complementary enzymes were fused, to create a self-sufficient bifunctional enzyme that can convert alcohols to esters or lactones. Three different ADH genes were fused to a gene coding for a thermostable CHMO, in both orientations (ADH-CHMO and CHMO-ADH). All six fusion enzymes could be produced and purified. For two of the three ADHs, we found a clear difference between the two orientations: one that showed the expected ADH activity, and one that showed low to no activity. The ADH activity of each fusion enzyme correlated with its oligomerization state. All fusions retained CHMO activity, and stability was hardly affected. The TbADH-TmCHMO fusion was selected to perform a cascade reaction, producing ε-caprolactone from cyclohexanol. By circumventing substrate and product inhibition, a > 99% conversion of 200 mM cyclohexanol could be achieved in 24 h, with > 13,000 turnovers per fusion enzyme molecule.

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. As such, NOXs are attractive pharmacological targets in immunomodulation, fibrosis and cancer. We have studied an extensive number of available NOX inhibitors, with the specific aim to identify bona fide ligands versus ROS-scavenging molecules. Accordingly, we have established a comprehensive platform of biochemical and biophysical assays. Most of the investigated small molecules revealed ROS-scavenging and/or assay-interfering properties to various degrees. A few compounds, however, were also demonstrated to directly engage one or more NOX enzymes. Diphenylene iodonium was found to react with the NOXs' flavin and heme prosthetic groups to form stable adducts. We also discovered that two compounds, VAS2870 and VAS3947, inhibit NOXs through the covalent alkylation of a cysteine residue. Importantly, the amino acid involved in covalent binding was found to reside in the dehydrogenase domain, where the nicotinamide ring of NADPH is bound. This work can serve as a springboard to guide further development of bona fide ligands with either agonistic or antagonistic properties toward NOXs.

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. Of the 177 selected mutations, 25 mutations brought about a significant increase in apparent melting temperature (ΔTm ≥ +3 °C). By combining mutations, a 10-fold mutant was generated with a Tm of 94 °C (+51 °C relative to wild type), almost reaching water's boiling point, and the highest increase with FRESCO to date. The 10-fold mutant's structure was elucidated, which enabled the identification of an activity-impairing mutation. After reverting this mutation, the enzyme showed no loss in activity compared to wild type, while displaying a Tm of 88 °C (+45 °C relative to wild type). This work demonstrates the value of enzyme stabilization through computational library design.