Corticosteroid Biosynthesis Revisited: Substrate Specificity of Steroid 21‐Hydroxylase

Objective Cytochrome P450 enzymes (CYPs) are capable of catalyzing regio‐ and stereo‐specific oxy functionalization reactions which otherwise are major challenges in organic chemistry. In order to make the best possible use of these biocatalysts it is imperative to understand their specificities. Human CYP21A2 (steroid 21‐hydroxylase) is known as one important enzyme in the biotransformation of progestins into corticosteroids. Humans with a reduced activity of steroid 21‐hydroxylase suffer from congenital adrenal hyperplasia (CAH), that is characterized by reduced production of cortisol. Due to feedback mechanisms of the hypothalamic pituitary adrenal axis, increased concentrations of progestins, but also androgens resulting from alternative metabolic pathways are observed in these patients. As enzymes in steroid biosynthesis are generally considered to show high substrate selectivity a closer investigation on structure requirements of steroid 21‐hydroxylase was conducted. Methods Biotransformation experiments were performed using a fission yeast expressing human CYP21A2 (CAD75) in a whole‐cell biotransformation assay . Progesterone, pregnenolone, and their corresponding 17‐hydroxy analogues were used as substrates. Product formation was monitored by GC‐MS. Molecular docking experiments were performed using GOLD software using the X‐ray structure of CYP21A2 co‐crystallized with progesterone (17, PDB entry 4Y8W ) as protein conformation. Results An investigation on A‐ring requirements yielded that the presence of a 3‐oxo group is a strict prerequisite for a CYP21A2 substrate . In line with these findings the incubation of the endogenous progestins pregnenolone (3β‐hydroxy‐pregn‐5‐en‐20‐one) and 17α‐hydroxypregnenolone (3β,17α‐dihydroxy‐pregn‐5‐en‐20‐one) did not result in successful 21‐hydroxylation, while progesterone (pregn‐4‐ene‐3,20‐dione) and 17‐hydroxyprogesterone (17α‐hydroxy‐pregn‐4‐ene‐3,20‐dione) were hydroxylated at C‐21. Molecular docking experiments supported the structure requirements. Apparently, the interaction of the carbonyl group at C‐3 of the substrate to the side‐chain Arg234 of the enzyme is indispensable. Conclusions The results are opposed to actual scientific view of steroid biosynthesis which for example supposes formation of 21‐hydroxypregnenolone is also catalyzed by CYP21A2 . They also help to understand metabolic pathways of steroids in the human body and may be useful in further synthesis experiments due to the use of enzymes as biocatalysts.