CYP7B, A NOVEL BRAIN CYTOCHROME-P450, CATALYZES THE SYNTHESIS OF NEUROSTEROIDS 7-ALPHA-HYDROXY DEHYDROEPIANDROSTERONE AND 7-ALPHA-HYDROXY PREGNENOLONE

Steroids produced locally in brain (neurosteroids), including dehydroe piandrosterone (DHEA), influence cognition and behavior. We previously described a novel cytochrome P450, Cyp7b, strongly expressed in rat a nd mouse brain, particularly in hippocampus, Cyp7b is most similar to steroidogenic P450s and potentially could play a role in neurosteroid metabolism To examine the catalytic activity of the enzyme mouse Cyp7b cDNA was introduced into a vaccinia virus vector, Extracts from cells infected with the recombinant showed NADPH-dependent conversion of DH EA (K-m, 13.6 mu M) and pregnenolone (K-m, 4.0 mu M) to slower migrati ng forms on thin layer chromatography, The expressed enzyme was less a ctive against 25-hydroxycholesterol, 17 beta-estradiol and 5 alpha-and rostane-3 beta,17 beta-diol, with low to undetectable activity against progesterone, corticosterone, and testosterone, On gas chromatography and mass spectrometry of the Cyp7b metabolite of DHEA the retention t ime and fragmentation patterns were identical to those obtained with a uthentic 7 alpha-hydroxy DHEA, The reaction product also comigrated on thin layer chromatography with 7 alpha-hydroxy DHEA but not with 7 be ta-hydroxy DHEA; when [7 alpha-H-3] pregnenolone was incubated with Cy p7b extracts the extent of release of radioactivity into the medium su ggested that hydroxylation was preferentially at the 7 alpha position. Brain extracts also efficiently liberated tritium from [7 alpha-H-3] pregnenolone and converted DHEA to a product with a chromatographic mo bility indistinguishable from 7 alpha-hydroxy DHEA. We conclude that C yp7b is a 7 alpha-hydroxylase participating in the synthesis, in brain , of neurosteroids 7 alpha-hydroxy DHEA, and 7 alpha-hydroxy pregnenol one.