Identification and characterization of the G15D mutation found in a male patient with 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) deficiency: alteration of the putative NAD-binding domain of type II 3 beta-HSD.

We report the detection of a homozygous G to A mutation converting codon Gly15 into Asp15 in the type II 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4-isomerase (3 beta-HSD) gene in a male pseudo-hermaphrodite born from consanguineous parents and suffering from severe salt-losing 3 beta-HSD deficiency. To investigate further the potential involvement of residue 15 in the beta alpha beta dinucleotide-binding fold, we have studied the effect of substituting Gly15 for Ala15. We assessed the effect of the G15D and G15A missense mutations on enzymatic activity by analyzing mutant enzymes generated by site-directed mutagenesis of type II 3 beta-HSD cDNA after their transient expression in COS-1 cells. In intact transfected cells, after a 2-h incubation, the percentage of conversion of [3H]pregnenolone (PREG) into [3H]progesterone (PROG) was 35% and 50% for the G15A and native type II 3 beta-HSD enzymes, respectively, whereas no detectable activity was observed in cells expressing the G15D protein. This finding is in agreement with the severity of the disease in the homozygote G15D index case. On the other hand, in homogenates from cells transfected with the normal pCMV-type II 3 beta-HSD plasmid or with the mutated pCMV-G15D or pCMV-G15A plasmid, the Km values for PREG were 0.72 microM, 3.2 microM, and 3.4 microM, respectively, when incubated for 1 h in the presence of excess (1 mM) NAD+. Moreover, the expressed G15D and G15A proteins had decreased affinities for NAD+ with Km values of 113 microM and 148 microM, respectively, compared with 22 microM for normal type II 3 beta-HSD.(ABSTRACT TRUNCATED AT 250 WORDS)