Abstract
The cDNA of a novel human glutathione transferase (GST) of the Alpha class was cloned, and the corresponding protein, denoted GST A3-3, was heterologously expressed and characterized. GST A3-3 was found to efficiently catalyze obligatory double-bond isomerizations of Delta(5)-androstene-3,17-dione and Delta(5)-pregnene-3,20-dione, precursors to testosterone and progesterone, respectively, in steroid hormone biosynthesis. The catalytic efficiency (k(cat)/K(m)) with Delta(5)-androstene-3,17-dione was determined as 5 x 10(6) m(-1) s(-1), which is considerably higher than with any other GST substrate tested. The rate of acceleration afforded by GST A3-3 is 6 x 10(8) based on the ratio between k(cat) and the rate constant for the nonenzymatic isomerization of Delta(5)-androstene-3,17-dione. Besides being high in absolute numbers, the k(cat)/K(m) value of GST A3-3 exceeds by a factor of approximately 230 that of 3beta-hydroxysteroid dehydrogenase/isomerase, the enzyme generally considered to catalyze the Delta(5)-Delta(4) double-bond isomerization. Furthermore, GSTA3-specific polymerase chain reaction analysis of cDNA libraries from various tissues showed a message only in those characterized by active steroid hormone biosynthesis, indicating a selective expression of GST A3-3 in these tissues. Based on this finding and the high activity with steroid substrates, we propose that GST A3-3 has evolved to catalyze isomerization reactions that contribute to the biosynthesis of steroid hormones.
Highlights
The metabolic pathways of steroid hormone biosynthesis leading to compounds such as testosterone and progesterone start with cholesterol and proceed in multiple steps involving oxidation and isomerization reactions [1]
Based on this finding and the high activity with steroid substrates, we propose that GST A3-3 has evolved to catalyze isomerization reactions that contribute to the biosynthesis of steroid hormones
Cloning of GSTA3 cDNA and Analysis of GST A3-3 Expression—cDNA libraries from various tissues were screened for GSTA3 expression using GSTA3-specific PCR
Summary
GSTA3 cDNA Cloning—Oligonucleotides that would amplify the cDNA of GST A3-3 were designed based on the sequence of the GSTA3 gene [10]. Because of the presence of many internal restriction sites in the GSTA3 cDNA, we eliminated an internal SalI site by introducing a silent mutation to be able to subclone the cDNA into the expression vector pKK-D [11] using EcoRI and SalI. This was accomplished by using inverted PCR, cloned Pfu DNA polymerase, and the 5Ј-end phosphorylated primers GSTA3327Rev (5Ј-GCAGAAGAAGGATCATTTCATTCAAAT-3Ј) and GSTA3328Forw (5Ј-CCTTATGTCGTCCTGAGAAAAAGAT-3Ј) and pGEMGSTA3 as template. Steady-state Kinetic Measurements—The isomerization activity of GST A3-3 with ⌬5-AD and ⌬5-PD was monitored both at pH 7.4 in PBS (37 °C) and pH 8.0 in 25 mM sodium phosphate buffer (30 °C) at the saturating 1 mM concentration of GSH. The Ki value of ⌬4-AD was determined by fitting the equation for competitive inhibition to the experimental data
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