Functional characterization of the novel L108W and P186L mutations detected in the type II 3?-hydroxysteroid dehydrogenase gene of a male pseudohermaphrodite with congenital adrenal hyperplasia

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Abstract
Two Isoenzymes are responsible for 3β-hydroxysterold dehydrogenase/Δ5-Δ4-isomerase (3β-HSD) activity in humans. We analyzed the structure of types I and II 3β-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 3β-HSD gene, but we found two different missense mutations in exon IV of the type II 3β-HSD gene of the patient; a conversion of codon Leu108 into a Trp (L108W) inherited from his mother and a conversion of codon Pro186 into a Leu (P186L) inherited from his father. We assessed the effect of the L108W and P186L mutations on 3β-HSD activity by in vitro analysis of mutant enzymes expressed in heterologous COS-1 cells. Using homogenates from transfected cells, the Km values for PREG were 7 ± 2 and 8 ± 2 μM for the recombinant L108W and P186L enzymes, respectively, compared with 2.2 ± 0.2 μM for the normal type II 3β-HSD enzyme. Moreover, Km values for NAD+ were much higher for the L108W and P186L proteins, being 678 ± 166 and 920 ± 351 μM, respectively, compared with 24 ± 3 μM for the normal type II 3β-HSD enzyme. Vmax 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 3β-HSD enzyme superfamily. Moreover, this patient is the first case presenting the salt-wasting form of classical 3β-HSD deficiency caused by mutated alleles possessing residual enzymatic activity. The combination of decreased Km values for the sterold substrate and cofactor thus explains the severe form of this enzymatic defect responsible for congenital adrenal hyperplasia and male pseudohermaphroditlsm.

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