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 HO. Recently, the Molecular Lego approach was applied to join the heme domain of CYP116B5 to sarcosine oxidase (SOX), which provides HO 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 T of 48.4 ± 0.04 and 58.3 ± 0.02°C and a enthalpy value ... More
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 HO. Recently, the Molecular Lego approach was applied to join the heme domain of CYP116B5 to sarcosine oxidase (SOX), which provides HO 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 T 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 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 HO (4.4 ± 0.8 and 11.0 ± 2.6% heme leakage respectively). Thanks to the in-situ HO generation, an improved k/K for the p-nitrophenol conversion was observed (k of 20.1 ± 0.6 min and K of 0.23 ± 0.03 mM), corresponding to 4 times the k/K 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 HO supply, equal to the 25% of the total drug conversion.
