CYP116B5-SOX: An artificial peroxygenase for drug metabolites production and bioremediation.

CYP116B5 is a class VII P450 in which the heme domain is linked to a FMN and 2Fe2S-binding reductase. Our laboratory has proved that the CYP116B5 heme domain (CYP116B5-hd) is capable of catalyzing the oxidation of substrates using H2O2. Recently, the Molecular Lego approach was applied to join the heme domain of CYP116B5 to sarcosine oxidase (SOX), which provides H2O2 in-situ by the sarcosine oxidation. In this work, the chimeric self-sufficient fusion enzyme CYP116B5-SOX was heterologously expressed, purified, and characterized for its functionality by absorbance and fluorescence spectroscopy. Differential scanning calorimetry (DSC) experiments revealed a TM of 48.4 ± 0.04 and 58.3 ± 0.02°C and a enthalpy value of 175,500 ± 1850 and 120,500 ± 1350 cal mol-1 for the CYP116B5 and SOX domains respectively. The fusion enzyme showed an outstanding chemical stability in presence of up to 200 mM sarcosine or 5 mM H2O2 (4.4 ± 0.8 and 11.0 ± 2.6% heme leakage respectively). Thanks to the in-situ H2O2 generation, an improved kcat/KM for the p-nitrophenol conversion was observed (kcat of 20.1 ± 0.6 min-1 and KM of 0.23 ± 0.03 mM), corresponding to 4 times the kcat/KM of the CYP116B5-hd. The aim of this work is the development of an engineered biocatalyst to be exploited in bioremediation. In order to tackle this challenge, an E. coli strain expressing CYP116B5-SOX was employed to exploit this biocatalyst for the oxidation of the wastewater contaminating-drug tamoxifen. Data show a 12-fold increase in tamoxifen N-oxide production-herein detected for the first time as CYP116B5 metabolite-compared to the direct H2O2 supply, equal to the 25% of the total drug conversion.

CYP116B5hd, a self-sufficient P450 cytochrome: A dataset of its electronic and geometrical properties.

This paper documents the dataset obtained from the Electron Paramagnetic Resonance (EPR) study of the electronic properties of a self-sufficient cytochrome P450, CYP116B5hd, which possesses an interesting catalytic activity for synthetic purposes. In fact, when isolated, its heme domain can act as a peroxygenase on different substrates of biotechnological interest. Raw data shown in Famulari et al. (2022) and supplementary data in raw and processed forms (figures) are documented and available in this paper. Additionally, simulations of the experimental data together with simulation scripts based for EasySpin, a widespread MATLAB toolbox for EPR spectral simulations, are provided. The procedure for g-value analysis based on a crystal-field theory is also detailed here, offering an interesting tool for comparison of FeIII-heme P450 systems. Due to the catalytic interest of the protein, which has been recently discovered, and the correlation that has been reported between g-values and peroxidase function, both, CW-EPR and HYSCORE spectra and data set of the model CYPBM3hd are also provided. Finally, the materials and methods for enzyme production and purification, sample preparation and experimental and spectroscopic procedures a together with instrumental details are described in detail. The data files and simulation scripts can be found in: https://doi.org/10.5281/zenodo.6418626.

Engineered human CYP2C9 and its main polymorphic variants for bioelectrochemical measurements of catalytic response.

Polymorphism is an important aspect in drug metabolism responsible for different individual response to drug dosage, often leading to adverse drug reactions. Here human CYP2C9 as well as its polymorphic variants CYP2C9*2 and CYP2C9*3 present in approximately 35% of the Caucasian population have been engineered by linking their gene to the one of D. vulgaris flavodoxin (FLD) that acts as regulator of the electron flow from the electrode surface to the haem. The redox properties of the immobilised proteins were investigated by cyclic voltammetry and electrocatalysis was measured in presence of the largely used anticoagulant drug S-warfarin, marker substrate for CYP2C9. Immobilisation of the CYP2C9-FLD, CYP2C9*2-FLD and CYP2C9*3-FLD on DDAB modified glassy carbon electrodes showed well defined redox couples on the oxygen-free cyclic voltammograms and mid-point potentials of all enzymes were calculated. Electrocatalysis in presence of substrate and quantification of the product formed showed lower catalytic activities for the CYP2C9*3-FLD (2.73 ± 1.07 min-1) and CYP2C9*2-FLD (12.42 ± 2.17 min-1) compared to the wild type CYP2C9-FLD (18.23 ± 1.29 min-1). These differences in activity among the CYP2C9 variants are in line with the reported literature data, and this set the basis for the use of the bio-electrode for the measurement of the different catalytic responses towards drugs very relevant in therapy.