CLONING AND EXPRESSION OF CDNA OF HUMAN DELTA(4)-3-OXOSTEROID 5-BETA-REDUCTASE AND SUBSTRATE-SPECIFICITY OF THE EXPRESSED ENZYME

The enzyme Delta(4)-3-oxosteroid 5 beta-reductase (3-oxo-5 beta-steroi d: NADP(+) oxidoreductase and 4,5 beta-dihydrocortisone: NADP(+) Delta 4-oxidoreductase) catalyzes the reduction of the Delta 4 double bond of bile acid intermediates and steroid hormones carrying the Delta(4)- 3-one structure in the A/B cis configuration. Human Delta(4)-3-oxoster oid 5 beta-reductase cDNA was isolated from a liver cDNA library by cr oss hybridization with a previously cloned rat cDNA, which was used as a probe [Onishi, Y., Noshiro, M., Shimosato, T and Okuda, K.-I. (1991 ) FEBS Lett. 283, 215-218]. DNA sequence analysis of a hybridization-p ositive clone predicted the human Delta(4)-3-oxosteroid SP-reductase t o contain 326 amino acids. The amino acid sequence of the human Delta( 4)-3-oxosteroid SP-reductase had 79% overall identity to the rat enzym e sequence. It also showed 54% and 50% overall identity with rat 3 alp ha-hydroxysteroid dehydrogenase and human aldose reductase, respective ly. RNA blotting analysis demonstrated the existence of a single Delta (4)-3-oxosteroid 5 beta-reductase mRNA of approximately 2.7 kb in huma n liver. Transfection of the cDNA into COS cells resulted in the expre ssion of an active enzyme with a high activity toward the bile acid in termediates 7 alpha 12 alpha-dihydroxy-4-cholesten-3-one and 7 alpha-h ydroxy-4-cholesten-3-one. In addition, the expressed enzyme showed a s mall but significant 5 beta-reduction activity toward 11 beta,17 alpha ,21-trihydroxy-Delta(4)-pregnene-3,20-dione (cortisol) and 17 beta-hyd roxy-Delta(4)-androsten-3-one (testosterone) whereas no activity was o bserved toward Delta(4)-pregnene-3,20-dione (progesterone) or Delta(4) -androstene-3-17-dione (androstenedione). The substrate specificity of the human enzyme is considerably narrower than that of the rat enzyme , and the enzyme seems to be more important for bile acid biosynthesis than for metabolism of steroid hormones.