Sensing the Generation of Intracellular Free Electrons Using the Inactive Catalytic Subunit of Cytochrome P450s as a Sink.

Damilare D Akintade,Bhabatosh Chaudhuri

doi:10.3390/s20144050

Damilare D Akintade, Bhabatosh Chaudhuri

Open Access

https://doi.org/10.3390/s20144050

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Abstract

Cytochrome P450 reductase (CPR) abstracts electrons from Nicotinamide adenine dinucleotide phosphate H (NADPH), transferring them to an active Cytochrome P450 (CYP) site to provide a functional CYP. In the present study, a yeast strain was genetically engineered to delete the endogenous CPR gene. A human CYP expressed in a CPR-null (yRD−) strain was inactive. It was queried if Bax—which induces apoptosis in yeast and human cells by generating reactive oxygen species (ROS)—substituted for the absence of CPR. Since Bax-generated ROS stems from an initial release of electrons, is it possible for these released electrons to be captured by an inactive CYP to make it active once again? In this study, yeast cells that did not contain any CPR activity (i.e., because the yeasts’ CPR gene was completely deleted) were used to show that (a) human CYPs produced within CPR-null (yRD-) yeast cells were inactive and (b) low levels of the pro-apoptotic human Bax protein could activate inactive human CYPs within this yeast cells. Surprisingly, Bax activated three inactive CYP proteins, confirming that it could compensate for CPR’s absence within yeast cells. These findings could be useful in research, development of bioassays, bioreactors, biosensors, and disease diagnosis, among others.

Highlights

cytochrome P450 (CYP450) catalyzes xenobiotic metabolism phase I reactions that mostly occur via oxidation reactions for both exogenous compounds and endogenous substrates [1]
CYP450 reductase (CPR)-null yRD− cells were selected on aureobasidin-containing YPD plates to continuously ensure that the yRD− cells were devoid of Initially, yeast strain lacked the endogenous yRD gene and was transformed with three episomal plasmids to the human CYP1A2 gene driven by (a) alcohol dehydrogenase 2 (ADH2), (b) glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and (c) PGK1 promoters, which had been isolated from the yeast
The results show that the CPR is essential expression is driven by the GAPDH promoter (GAPDHp)

Summary

Introduction

CYP450 catalyzes xenobiotic metabolism phase I reactions that mostly occur via oxidation reactions for both exogenous compounds and endogenous substrates [1]. Functional cytochrome P450 (CYP450) proteins are enzymes that take part in a diverse number of biochemical reactions in different organisms [2]. CYP450 (CYP) enzymes are present in all organisms and belong to a superfamily of proteins [5]. They are the main catalyst involved in the biotransformation of a wide range of chemical compounds that act as substrates to the CYP450 enzymes. The biotransformation mediated by CYP450 enzymes occurs through oxidation. One of the reactions that CYP450 enzymes perform on their substrates is the hydroxylation of unreactive carbon atoms [4,6,7]

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Discussion

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