Mutational analysis of SRD5A2: From gene to functional kinetics in individuals with steroid 5α-reductase 2 deficiency.

Human steroid 5α-reductase 2 (SRD5A2) plays a determinative role in the masculinization of external genitalia. To date, approximately 114 different mutations of the SRD5A2 gene have been reported; however, little information is available about their impact on catalytic function or their three-dimensional (3D) structures. We determined the effect of point mutations on the testosterone-depend kinetic constants (K _m,app and V _max,app ) and structural characteristics of SRD5A2 from Mexican patients with 46,XY-steroid 5α-reductase 2 deficiency. PCR-SSCP assays identified ten distinct gene variants and sequencing analysis identified missense mutations [p.V3I, p.S14R, p.A52T, p.F118L, p.R145W, p.R171S, p.L226P, p.F229S, p.S245Y, and p.A248V]. Mutations were re-created by site-directed mutagenesis and expressed in HEK293 cells. Functional studies demonstrated that 8 variants led to partial (K _m,app = 0.16-2.6 μM; V _max,app = 224-2640 pmol/mg P/min) or complete losses of activity compared to the wild-type enzyme (K _m,app = 0.7 μM; V _max,app = 4044 pmol/mg P/min). All the mutations were assessed using multiple software tools and the results predicted that all of the mutations were associated with disease or damage. Mapping mutations on the model of a 3D structure of SRD5A2 demonstrated alterations in contact sites with their proximal amino acids. Our data show that mutations affect the catalytic efficiency (V _max /K _m ) or result in residual enzymatic activity, which could be due to erroneous interactions between amino acid residues, the substrate testosterone, or NADPH.
Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest.
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