FUNCTIONAL-CHARACTERIZATION OF THE NOVEL L108W AND P186L MUTATIONS DETECTED IN THE TYPE-II 3-BETA-HYDROXYSTEROID DEHYDROGENASE GENE OF A MALE PSEUDOHERMAPHRODITE WITH CONGENITAL ADRENAL-HYPERPLASIA

Two isoenzymes are responsible for 3 beta-hydroxysteroid dehydrogenase/Delta(5)-Delta(4)-isomerase (3 beta-HSD) activity in humans. We analyzed the structure of types I and II 3 beta-HSD genes in a male pseudohermaphrodite suffering from a severe salt-losing form of congenital adrenal hyperplasia. We did not detect any mutation in the type I 3 beta-HSD gene, but we found two different missense mutations in exon IV of the type II 3 beta-HSD gene of the patient; a conversion of codon Leu(108) into a Trp (L108W) inherited from his mother and a conversion of codon Pro(186) into a Leu (P186L) inherited from his father. We assessed the effect of the L108W and P186L mutations on 3 beta-HSD activity by in vitro analysis of mutant enzymes expressed in heterologous COS-1 cells. Using homogenates from transfected cells, the K-m values for PREG were 7 +/- 2 and 8 +/- 2 mu M for the recombinant L108W and P186L enzymes, respectively, compared with 2.2 +/- 0.2 mu M for the normal type II 3 beta-HSD enzyme. Moreover, K-m values for NAD(+) were much higher for the L108W and P186L proteins, being 678 +/- 166 and 920 +/- 361 mu M, respectively, compared with 24 +/- 3 mu M for the normal type II BP-HSD enzyme. V-max values for PREG and NAD(+) were lower for both mutant enzymes; thus, the in vitro overall efficiency, relative to the normal enzyme, is approximate as 0.3% and 0.2% for the L108W and P186L enzymes, respectively. The present study is the first description of mutations which significantly affect the affinity for NAD(+) in addition to reducing the affinity for PREG, thus providing useful information on the structure-activity relationships of the 3 beta-HSD enzyme superfamily. Moreover, this patient is the first case presenting the salt-wasting form of classical 3 beta-HSD deficiency caused by mutated alleles possessing residual enzymatic activity. The combination of decreased K-m values for the steroid substrate and cofactor thus explains the severe form of this enzymatic defect responsible for congenital adrenal hyperplasia and male pseudohermaphroditism.