P450 Oxidoreductase Deficiency: loss of activity caused by protein instability from a novel L374H mutation.

Parween, Shaheena; Boulez, Florence Roucher; Flück Pandey, Christa Emma; Lienhardt-Roussie, Anne; Mallet, Delphine; Morel, Yves; Pandey, Amit Vikram (2016). P450 Oxidoreductase Deficiency: loss of activity caused by protein instability from a novel L374H mutation. The Journal of clinical endocrinology and metabolism, 101(12), jc.2016-1928. The Endocrine Society 10.1210/jc.2016-1928

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CONTEXT

P450 oxidoreductase (POR) is required for the activities of steroid metabolizing cytochrome P450 enzymes in the endoplasmic reticulum. P450 oxidoreductase deficiency (PORD) is a form of congenital adrenal hyperplasia.

OBJECTIVE AND AIM

Enzymatic and structural analysis of a novel L374H POR mutation from a patient with 46, XX disorder of sexual development. Design, setting, patient and intervention: 46,XX girl with non-consanguineous Turkish parents. Virilized external genitalia at birth; uterus and ovaries present, no sign of Antley-Bixler syndrome. Initial diagnosis of CYP21A2 deficiency, no mutations in CYP21A2 but found POR mutations. Functional testing done after producing recombinant POR proteins for analyzing enzymatic and structural properties.

MAIN OUTCOME

Novel mutations causing severe loss of POR activities for metabolism of steroids and small molecules.

RESULTS

The L374H reduced activities by 80% in cytochrome c, 97% in MTT and 86% in ferricyanide reduction assays. The catalytic efficiency of 17 α-hydroxylation of progesterone and 17, 20-lyase reaction of 17-OH pregnenolone was decreased by 87% and 90 %, 21-hydroxylation of progesterone was decreased by 96 % and androstenedione aromatization was decreased by 90%. Analysis of mutant structure by molecular dynamic simulations revealed structure instability. Flavin release and fast proteolysis assays showed that the L374H mutant is less stable than WT POR, confirming bioinformatics prediction.

CONCLUSIONS

This is the first report of a mutation causing PORD by affecting protein stability which causes severe reduction in POR activities. Detailed characterization of individual mutations in POR is required for understanding novel molecular mechanisms causing PORD.

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