Structure and Biochemical Properties of the 5aReductase Isozymes

The existence of more than one 5a-R isozymes had been postulated years ago on the basis of studies utilizing various inhibitors (99,100), and different substrates (99). More recently, two isoforms of the 5 a-R (called type 1 and type 2) have been cloned in humans, rat, and monkey (101-106). In humans, the type 1 5a-R gene is composed of five exons and four introns and produces a protein of 259 amino acids. The type 2 5a-R gene has a similar structure, but the resulting protein is composed of254 aminoacids. The structures of the enzymatic proteins, determined from their respective cDNAs, show a limited degree of homology (about 47%), and a predicted molecular weight of 28-29 kDa. The homology between the human and rat enzymes is 60% for the type 1, and 77% for the type 2 isozyme. The monkey isoforms are structurally and functionally more similar to their respective human counterparts. In terms of primary structures, both isozymes are composed of a high number of hydrophobic aminoacid residues, distributed throughout the molecule; this strongly imposes the localization of the enzymes in cell membranes (see this section). Consensus sequences for the binding of substrates similar to those present in other reducing enzymes have not been found in either isozyme, but mutational analysis has permitted to localize the region of substrate binding in the N-terminal portion of the protein, which is encoded by the first exon; the binding site for NADPH, the cofactor of the 5a-R, appears to be located in the C-terminal portion of the protein, a region encoded by the 4th and 5th exon. However, the exact location of both domains has not been defined, because amino acids relevant for the maintenance of a correct affinity for the substrate have been identified also in the C-terminal region of the molecule (105). Heterogeneity of the rat type 1 enzyme has been recently described and a cDNA coding for a 5a-R type 1b with four additional amino acids in the amino terminal region has been cloned (107). This protein shows a higher affinity for the various substrates than the shorter one, particularly in the case of cortisol for which the Km is decreased 7.5 fold; the decrease observed in the case of T and PROG is only 4.5-fold.

Despite the fact that the two major isoforms of the 5a-R (types 1 and 2) catalyze the same reaction (e.g., T to 5a-DHT, PROG to 5a-DHT, and so forth), they possess different biochemical and possibly functional properties.

In rats, the affinity of T for the type 1 isoform is about 15-20-fold lower than that determined for the type 2 isoform. The difference in affinity is still evident in the case of the human enzymes, but is less marked. Both in rat and in humans, the capability of reducing the substrate is much higher in the case of the type 1 isoform.

The two isoforms have a different pH optimum: the type 1 isoform is active in a wide range of pH (from 5-8), whereas the type 2 5a-R possesses a narrow pH optimum around 5, with a very low activity at pH 7.5. The two isozymes show also a differential sensitivity to synthetic inhibitors designed, like finasteride, to block the human type 2 isozyme (108), as well as to those not specifically designed for this isozyme (109). Finasteride interacts with four amino acid residues present in the N-terminal region of the protein (108). Little is known on the efficacy of "selective" 5a-R type 1 inhibitors on the rat and human type 2 isozymes.

The human type 1 gene (gene symbol SRD5A1) is located at the extreme tip of the short arm (p15) of chromosome 5. An apparently nonfunctional pseudogene, lacking introns and containing a premature translation termination codon, is present on the long arm of the X chromosome. The human type 2 gene (gene symbol SRD5A2) is present on the short arm of chromosome 2. The exon-intron structures of the two functional genes is identical, indicating an ancient gene duplication. In humans, the type 1 gene appears to be expressed at high levels in the liver and in the nongenital skin, and at low levels in the androgen target tissues (105). In the rat, the expression of this isoform is widely distributed throughout the various genital and nongenital tissues, with the highest levels in the liver. Both in rat and in humans, the type 2 isoform is concentrated especially in andro-gen-dependent structures, such as the epididymis (especially in the basal cells), the seminal vesicles, the genital skin, and the prostate, where it is mostly present in the stromal component. Only small amounts of 5a-R type 2 have been detected in other tissues. On the contrary, the type 1 isoform appears to be mainly located in the prostatic epithelial cells (105,110).

There is no doubt that, in the prostate, a structure that contains both the type 1 and type 2 isozymes, the most relevant isozyme is represented by the type 2 isoform. The presence in human of a genetically determined ineffective type 2 isoform produces a syndrome of male pseudohermaphroditism (named after Imperato-McGinley) characterized by aplasia of the prostate and ambiguity of the external genitalia (111,112). It is obvious that, in this pathological condition, the presence of a normal type 1 isozyme is unable to counteract the consequences of the abnormality of the type 2 isoform. No genetic deficiency syndromes of the type 1 isozyme have been described up to now in humans. In transgenic mice showing a 5a-R type 1 deficiency, a significant decrease in the number of live fetuses has been observed; this is apparently owing to an increased formation of estrogens, because the phenomenon can be reversed by antiestrogens administered to the mothers (97,98).

Several studies have been performed to determine the subcellular localization of the 5a-Rs, but the results are not very consistent. However, in all cases, the enzyme appears to be strongly associated to cellular membranes (cytoplasmic or nuclear). A possible different subcellular localization of the two isozymes has been proposed by fractionation studies performed, in the authors' laboratory, on yeast cells genetically transformed to specifically produce the two rat isozymes. The data have shown that the type 1 isozyme is associated to the nuclei, whereas the type 2 isozyme is present in the microsomal fractions (113).

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