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 cod on Gly(15) into Asp(15) in the type LT 3 beta-hydroxysteroid dehydroge nase/Delta(5)-Delta(4)-isomerase (3 beta-HSD) gene in a male pseudoher maphrodite born from consanguineous parents and suffering from severe salt-losing 3 beta-HSD deficiency. To investigate further the potentia l involvement of residue 15 in the beta alpha beta dinucleotide-bindin g fold, we have studied the effect of substituting Gly(15) for Ala(15) . We assessed the effect of the G15D and G15A missense mutations on en zymatic activity by analyzing mutant enzymes generated by site-directe d mutagenesis of type II 3 beta-HSD cDNA after their transient express ion in COS-1 cells. In intact transfected cells, after a 2-h incubatio n, the percentage of conversion of [H-3]pregnenolone (PREG) into [H-3] progesterone (FROG) was 35% and 50% for the G15A and native type II 3 beta-HSD enzymes, respectively, whereas no detectable activity was obs erved in cells expressing the G15D protein. This finding is in agreeme nt with the severity of the disease in the homozygote G15D index case. On the other hand, in homogenates from cells transfected with the nor mal pCMV-type II 3 beta-HSD plasmid or with the mutated pCMV-G15D or p CMV-G15A plasmid, the K-m values for PREG were 0.72 mu M, 3.2 mu M, an d 3.4 mu M, respectively, when incubated for 1 h in the presence of ex cess (1 mM) NAD(+). Moreover, the expressed G15D and G15A proteins had decreased affinities for NAD(+) with K-m values of 113 mu M and 148 m u M, respectively, compared with 22 mu M for normal type II 3 beta-HSD . The marked difference in activity of the G15D and G15A proteins expr essed in intact cells compared with their similar level of activity in homogenates remains to be elucidated, It could be suggested that, in addition to decreasing apparent affinity for both the substrate and co factor, the G15D mutation may alter the proper intracellular localizat ion of this integral membrane protein or that its association with int act membranes in vivo may exert some strain, preventing the adoption o f its final maximally efficient conformation.