Membrane topology and search for potential redox partners of colon cancer‐specific cytochrome P450 2W1

Abstract

Edited by Peter BrzezinskiCytochrome P450 2W1 (CYP2W1) is a colon tumor-specific enzyme, suggested as a potential target for cancer therapy. In contrast to other endoplasmic reticulum P450s, we found completely inverted ER membrane topology of CYP2W1 using different approaches (redox sensitive luciferase assay and protease protection assay) and demonstrated that canonical CYP reductants, cytochrome P450 reductase, and cytochrome b 5 cannot serve as electron donors for CYP2W1. Moreover, the reduced catalytic activity of the Asn177 mutant that is modified by glycan moieties in the wild-type enzyme indicates a functional relevance of CYP2W1 glycosylation.

The CYP2W1 enzyme: regulation, properties and activation of prodrugs

“…chain impossible. This was confirmed using cells with a knocked down expression of POR and inhibitors of POR and NADH cytochrome b 5 reductase (Guo et al, 2016). The intracellular CYP2W1 mediated metabolism of chloromethylindoline substrates in, for example intact cells indicates a functional electron transport chain with a specific electron donor for CYP2W1.…”

“…Mass spectrometry analysis confirmed glycosylation of Asn177 and mutation of this residue led to the disappearance of the 54 kDa band (Gomez et al, 2010). The Asn177 residue was found to be important for CYP2W1 catalytic activity (Guo et al, 2016) but still the role of the glycosylation for CYP2W1 function remains to be established.…”

“…However, (i) results from protease protection and (ii) results obtained based on expression of CYP2W1 as a fusion protein with a redox-sensitive luciferase that acquires full activity only upon oxidative folding characteristic for the ER lumen, demonstrated that CYP2W1 is indeed atypically localized (Figure 2) (Gomez et al, 2010;Guo et al, 2016). A subfraction of CYP proteins can be inversely incorporated into the ER to some minor extent; however, the predominant luminal orientation of CYP2W1 is truly unique.…”

“…The intracellular CYP2W1 mediated metabolism of chloromethylindoline substrates in, for example intact cells indicates a functional electron transport chain with a specific electron donor for CYP2W1. NADPH can be produced in the ER lumen and there are indications that CYP2W1 might be a terminal recipient of electrons from such luminally generated NADPH (Guo et al, 2016). Thus, the enigmatic redox partner(s) of CYP2W1 should be searched among various ER luminal oxidoreductases.…”

Understanding the Mechanisms Involved in the Regulation of Cytochrome P450 Gene Expression in Drosophila melanogaster (Diptera: Drosophilidae)

“…The cytochrome P450 superfamily consists of a considerable amount of heme-containing monooxygenases and is established in all living organisms including insects [1,2]. In the fruit fly, Drosophila melanogaster alone there are 83 P450 genes with a few of these enzymes playing key roles in the metabolism or activation of xenobiotics [3][4][5][6].…”