Biochemical Investigation of Membrane-Bound Cytochrome b5 and the Catalytic Domain of Cytochrome b5 Reductase from Arabidopsis thaliana

Iqbal, Tabish and Das, Debasis (2022) Biochemical Investigation of Membrane-Bound Cytochrome b5 and the Catalytic Domain of Cytochrome b5 Reductase from Arabidopsis thaliana. In: Biochemistry . ISSN 0006-2960

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Abstract

The endoplasmic reticulum (ER) membrane of plant cells contains several enzymes responsible for the biosynthesis of a diverse range of molecules essential for plant growth and holds potential for industrial applications. Many of these enzymes are dependent on electron transfer proteins to sustain their catalytic cycles. In plants, two crucial ER-bound electron transfer proteins are cytochrome b5 and cytochrome b5 reductase, which catalyze the stepwise transfer of electrons from NADH to redox enzymes such as fatty acid desaturases, cytochrome P450s, and plant aldehyde decarbonylase. Despite the high significance of plant cytochrome b5 and cytochrome b5 reductase, they have eluded detailed characterization to date. Here, we overexpressed the full-length membrane-bound cytochrome b5 isoform B from the model plant Arabidopsis thaliana in Escherichia coli, purified the protein employing detergents as well as styrene–maleic acid (SMA) copolymers, and biochemically characterized the protein. The SMA-encapsulated cytochrome b5 exhibits a discoidal shape and the characteristic features of the active heme-bound state. We also overexpressed and purified the soluble domain of cytochrome b5 reductase from A. thaliana, establishing its activity, stability, and kinetic parameters. Further, we demonstrated that the plant cytochrome b5, purified in detergents and styrene maleic acid lipid particles (SMALPs), readily accepts electrons from the cognate plant cytochrome b5 reductase and distant electron mediators such as plant NADPH-cytochrome P450 oxidoreductase and cyanobacterial NADPH-ferredoxin reductase. We also measured the kinetic parameters of cytochrome b5 reductase for cytochrome b5. Our studies are the first to report the purification and detailed biochemical characterization of the plant cytochrome b5 and cytochrome b5 reductase from the bacterial overexpression system.

Item Type:	Journal Article
Publication:	Biochemistry
Additional Information:	The copyright of this article belongs to American Chemical Society
Keywords:	Engineering controlled terms Biochemistry; Enzymes; Escherichia coli; Fatty acids; Kinetic parameters; Soaps (detergents); Styrene Engineering uncontrolled terms Arabidopsis thaliana; Catalytic domains; Cytochrome b5; Diverse range; Electron transfer proteins; Endoplasmic reticulum membranes; Kinetics parameter; Maleic acids; Membrane-bound; Plants cells Engineering main heading Purification
Department/Centre:	Division of Chemical Sciences > Inorganic & Physical Chemistry
Date Deposited:	23 May 2022 11:45
Last Modified:	23 May 2022 11:45
URI:	https://eprints.iisc.ac.in/id/eprint/72396

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