Natural Mutagenesis Study of the Human Steroid 5.alpha.-Reductase 2 Isoenzyme

Abstract

The enzyme steroid 5 alpha-reductase utilizes NADPH to reduce the double bonds of a variety of steroid substrates with generalized 3-oxo, delta 4,5 structures. One substrate for this membrane-bound enzyme is testosterone, whose reduction to dihydrotestosterone is required for the embryonic differentiation of the external male genitalia and prostate. There are two 5 alpha-reductase isozymes, designated types 1 and 2, which have different biochemical and pharmacological properties. Inherited deficiencies of 5 alpha-reductase type 2 result in a form of male pseudohermaphroditism in which the external genitalia fail to develop normally. Here, nine additional mutations in the 5 alpha-reductase 2 gene in subjects with 5 alpha-reductase deficiency are described. The biochemical consequences of these mutations, as well as 13 previously identified missense mutations, were characterized by recreating the mutations in an expressible cDNA and transfecting into mammalian cells. Twelve of the 22 mutations inactivated the enzyme. The remaining 10 mutations impaired enzyme function by affecting either substrate or cofactor binding. Almost all mutations decreased the half-life of the protein, and all but one of the impaired enzymes had an altered pH optimum. The mutations provide insight into functional domains in the protein as well as an unusual acidic pH optimum characteristic of the 5 alpha-reductase type 2 isozyme.

Identification and characterization of somatic steroid 5α-reductase (SRD5A2) mutations in human prostate cancer tissue

“…However, there appears to be a clustering of missense substitutions particularly in the carboxy-terminal third of the protein, specifically codons 183-248 (Figure 1). This part of the steroid 5a-reductase enzyme has been proposed to be involved in substrate and cofactor binding (Wigley et al, 1994;Makridakis et al, 2000). We also note the relative rarity of substitutions of any kind in the central portion of the SRD5A2 protein, another observation previously made by us but not others (Wigley et al, 1994;Makridakis et al, 2000).…”

“…This part of the steroid 5a-reductase enzyme has been proposed to be involved in substrate and cofactor binding (Wigley et al, 1994;Makridakis et al, 2000). We also note the relative rarity of substitutions of any kind in the central portion of the SRD5A2 protein, another observation previously made by us but not others (Wigley et al, 1994;Makridakis et al, 2000). We suspect that this difference may be due to the different phenotypes investigated by Russell and co-workers, male pseudohermaphroditism (cf.…”

“…We suspect that this difference may be due to the different phenotypes investigated by Russell and co-workers, male pseudohermaphroditism (cf. Wigley et al, 1994) a rare autosomal recessive disorder which results in mutations that severely impair enzymatic activity, and us who are interested in mutations and polymorphisms in prostate cancer that presumably activate steroid 5a-reductase (e.g. Makridakis et al, 1999Makridakis et al, , 2000.…”

“…Makridakis et al, 1999Makridakis et al, , 2000. Thus, male pseudohermaphrodistism mutations are loss of function mutations (Wigley et al, 1994) while activating mutations in prostate cancer should be gain of function mutations (Makridakis et al, 1999). Analysis of the apparent binding constants for the distinct missense substitutions (Table 3) can provide information about the number of domains that comprise the binding pocket of the prostatic steroid 5a-reductase, a protein that has never been purified.…”

“…Thus, this report is the first to systematically investigate the entire coding sequence of an androgen-metabolic gene for somatic mutations in prostate cancer. We propose that these genes are likely to be important targets in tumor progression since the disease is androgen-dependent at least early in its (Wigley et al, 1994;Makridakis et al, 2000). The positions of amino acids 100 and 200 are indicated as reference points Somatic SRD5A2 mutations in prostate cancer N Makridakis et al development (Cheng et al, 1993).…”