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.

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.

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.