title: Biochemical analyses and molecular modeling explain the functional loss of 17β-hydroxysteroid dehydrogenase 3 mutant G133R in three Tunisian patients with 46, XY Disorders of Sex Development.
abstract: Mutations in the HSD17B3 gene resulting in 17β-hydroxysteroid dehydrogenase type 3 (17β-HSD3) deficiency cause 46, XY Disorders of Sex Development (46, XY DSD). Approximately 40 different mutations in HSD17B3 have been reported; only few mutant enzymes have been mechanistically investigated. Here, we report novel compound heterozygous mutations in HSD17B3, composed of the nonsense mutation C206X and the missense mutation G133R, in three Tunisian patients from two non-consanguineous families. Mutants C206X and G133R were constructed by site-directed mutagenesis and expressed in HEK-293 cells. The truncated C206X enzyme, lacking part of the substrate binding pocket, was moderately expressed and completely lost its enzymatic activity. Wild-type 17β-HSD3 and mutant G133R showed comparable expression levels and intracellular localization. The conversion of Δ4-androstene-3,17-dione (androstenedione) to testosterone was almost completely abolished for mutant G133R compared with wild-type 17β-HSD3. To obtain further mechanistic insight, G133 was mutated to alanine, phenylalanine and glutamine. G133Q and G133F were almost completely inactive, whereas G133A displayed about 70% of wild-type activity. Sequence analysis revealed that G133 on 17β-HSD3 is located in a motif highly conserved in 17β-HSDs and other short-chain dehydrogenase/reductase (SDR) enzymes. A homology model of 17β-HSD3 predicted that arginine or any other bulky residue at position 133 causes steric hindrance of cofactor NADPH binding, whereas substrate binding seems to be unaffected. The results indicate an essential role of G133 in the arrangement of the cofactor binding pocket, thus explaining the loss-of-function of 17β-HSD3 mutant G133R in the patients investigated.
authors.affiliation: Division of Molecular and Systems Toxicology, Department of Pharmaceutical Sciences, Pharmacenter, University of Basel, Basel, Switzerland.
authors.forename: Roger T
authors.initials: RT
authors.lastname: Engeli
authors.affiliation: Human Molecular Genetics Laboratory, Faculty of Medicine, University of Sfax, Sfax, Tunisia.
authors.forename: Bochra Ben
authors.initials: BB
authors.lastname: Rhouma
authors.affiliation: Molecular Modeling, Department of Pharmaceutical Sciences, Pharmacenter, University of Basel, Basel, Switzerland.
authors.forename: Christoph P
authors.initials: CP
authors.lastname: Sager
authors.affiliation: Division of Molecular and Systems Toxicology, Department of Pharmaceutical Sciences, Pharmacenter, University of Basel, Basel, Switzerland.
authors.forename: Maria
authors.initials: M
authors.lastname: Tsachaki
authors.affiliation: Division of Molecular and Systems Toxicology, Department of Pharmaceutical Sciences, Pharmacenter, University of Basel, Basel, Switzerland.
authors.forename: Julia
authors.initials: J
authors.lastname: Birk
authors.affiliation: Human Molecular Genetics Laboratory, Faculty of Medicine, University of Sfax, Sfax, Tunisia.
authors.forename: Faiza
authors.initials: F
authors.lastname: Fakhfakh
authors.affiliation: Human Molecular Genetics Laboratory, Faculty of Medicine, University of Sfax, Sfax, Tunisia.
authors.forename: Leila
authors.initials: L
authors.lastname: Keskes
authors.affiliation: Human Molecular Genetics Laboratory, Faculty of Medicine, University of Sfax, Sfax, Tunisia; Department of Medical Genetics, HediChaker Hospital, Sfax, Tunisia. Electronic address: Neila.Belguith@fmsf.rnu.tn.
authors.forename: Neila
authors.initials: N
authors.lastname: Belguith
authors.affiliation: Division of Molecular and Systems Toxicology, Department of Pharmaceutical Sciences, Pharmacenter, University of Basel, Basel, Switzerland. Electronic address: alex.odermatt@unibas.ch.
authors.forename: Alex
authors.initials: A
authors.lastname: Odermatt
mesh_terms: D015067:17-Hydroxysteroid Dehydrogenases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D000293:Adolescent; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D002648:Child; D017124:Conserved Sequence; D058490:Disorder of Sex Development, 46,XY / Q000235:genetics*; D004721:Endoplasmic Reticulum / Q000378:metabolism; D005260:Female; D057809:HEK293 Cells; D006801:Humans; D008297:Male; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009249:NADP / Q000378:metabolism
chemical_list: D009249:NADP; D015067:17-Hydroxysteroid Dehydrogenases; C476565:17beta-hydroxysteroid dehydrogenase type 3
keywords: 17β-hydroxysteroid dehydrogenase; 46, XY DSD; HSD17B3; Male sexual development; Mutation; Structure function relationship; Testosterone