Characterization of rat 5α-reductases type 1 and type 2 expressed in Saccharomyces cerevisiae

Abstract

Two isoforms of the rat 5α-reductase (5α-R), the enzyme that converts testosterone into dihydrotestosterone (DHT), and other Δ4-3-keto steroids (e.g. progesterone and corticoids) into their 5α-reduced metabolites, have been cloned. In this study, a convenient and efficient system was developed to overexpress the two isoenzymes in Saccharomyces cerevisiae by using the ubiquitin-fusion expression system. Two yeast expression vectors have been prepared, YEpR1 and YEpR2, which code for 5α-R type 1 and 5α-R type 2 respectively; they contain the copper-responsive yeast metallothionein promoter (CUP1) upstream of the ubiquitin coding sequence, and the full-length rat 5α-R type 1 or 5α-R type 2 cDNAs in frame to the 3′ end of the ubiquitin cDNA. The activity of the two isoenzymes produced in yeast was determined in cell lysates at the enzyme pH optima (type 1, pH 7.5; type 2, pH 5.5) and a possible differential intracellular distribution was also evaluated. The kinetic parameters were: type 1, Km 4.6 μM, Vmax. 100.6 μg/h per mg of protein ; type 2, Km 68.6 nM, Vmax. 0.84 μg/h per mg of protein. Yeast cell lysates were fractionated by differential centrifugation and the 5α-R type 1 activity was maximal in fractions containing nuclei (1000 g and 2500 g), whereas the maximal activity of 5α-R type 2 was present in subcellular fractions sedimenting at higher speeds (20000 g). The data indicate that yeasts overexpress the two 5α-R isoenzymes, maintaining their native biochemical properties, and that the two isoforms are probably differentially localized within the yeast cell.