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 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 3 beta-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 microM for the recombinant L108W and P186L enzymes, respectively, compared with 2.2 +/- 0.2 microM for the normal type II 3 beta-HSD enzyme. Moreover, Km values for NAD+ were much higher for the L108W and P186L proteins, being 678 +/- 166 and 920 +/- 351 microM, respectively, compared with 24 +/- 3 microM for the normal type II 3 beta-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.(ABSTRACT TRUNCATED AT 250 WORDS)
Sanchez
R
R
Medical Research Council Group in Molecular Endocrinology, CHUL Research Center, Québec, Canada.
Mébarki
F
F
Rhéaume
E
E
Laflamme
N
N
Forest
M G
MG
Bey-Omard
F
F
David
M
M
Morel
Y
Y
Labrie
F
F
Simard
J
J
D015096:3-Hydroxysteroid Dehydrogenases / Q000235:genetics* / Q000378:metabolism; D000312:Adrenal Hyperplasia, Congenital / Q000150:complications / Q000201:enzymology* / Q000235:genetics*; D000595:Amino Acid Sequence; D000818:Animals; D017931:DNA Primers / Q000235:genetics; D012734:Disorders of Sex Development / Q000150:complications / Q000201:enzymology* / Q000235:genetics*; D005260:Female; D006801:Humans; D007231:Infant, Newborn; D007527:Isoenzymes / Q000235:genetics; D007700:Kinetics; D008297:Male; D008969:Molecular Sequence Data; D009154:Mutation; D010375:Pedigree; D016133:Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity
D017931:DNA Primers; D007527:Isoenzymes; D015096:3-Hydroxysteroid Dehydrogenases