Loss of protein stability and function caused by a single point mutation (P228L) in the Cytochrome P450 Oxidoreductase.

Rojas Velazquez, Maria Natalia; Noebauer, Mathias; Pandey, Amit V (2022). Loss of protein stability and function caused by a single point mutation (P228L) in the Cytochrome P450 Oxidoreductase. FASEB journal, 36(S1) Federation of American Societies for Experimental Biology 10.1096/fasebj.2022.36.S1.R5860

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Cytochrome P450 oxidoreductase (POR) is the obligatory redox partner of steroid and drug metabolizing cytochrome P450s located in the endoplasmic reticulum. Mutations in POR cause a broad range of disorders like congenital adrenal hyperplasia. Genome sequencing studies have revealed the existence of a POR missense variant P228L which was linked with reduced function of some P450 enzymes. We aimed to expand the enzymatic studies of POR variant P228L for its role in human metabolism. We expressed human wild type and the P228L variant POR in bacteria and purified the proteins by Affinity Chromatography. We tested the stability of the proteins using fast proteolysis and performed kinetics assays of POR activities using small molecules substrates. POR (WT or P228L) were mixed with purified cytochrome P450 proteins and activities of cytochrome P450 proteins were assayed. The single point mutation P228L had reduced thermal stability indicated by a lower melting point comparing to the WT. In the kinetics studies, the rates of the reactions with P228L were considerably lower than the WT but the Km did not show substantial changes. We observed a decrease in the enzymatic activities of CYP3A5 and CYP2C9 of more than 40% with P228L form of POR comparing to WT. A single change in the amino acid sequence can affect the protein stability and cause a severe reduction in POR activity. Molecular characterization of POR mutations is crucial to have a better understanding of the impact on the functionality of its redox Partners.

Item Type:	Journal Article (Further Contribution)
Division/Institute:	04 Faculty of Medicine > Pre-clinic Human Medicine > BioMedical Research (DBMR)
UniBE Contributor:	Pandey, Amit Vikram
Subjects:	600 Technology > 610 Medicine & health
ISSN:	1530-6860
Publisher:	Federation of American Societies for Experimental Biology
Language:	English
Submitter:	Pubmed Import
Date Deposited:	16 May 2022 13:40
Last Modified:	05 Dec 2022 16:19
Publisher DOI:	10.1096/fasebj.2022.36.S1.R5860
URI:	https://boris.unibe.ch/id/eprint/170018