The Journal of Steroid Biochemistry and Molecular Biology
3α/β,20β-Hydroxysteroid dehydrogenase (porcine testicular carbonyl reductase) also has a cysteine residue that is involved in binding of cofactor NADPH
Introduction
Porcine 3α/β,20β-hydroxysteroid dehydrogenase (3α/β,20β-HSD), also known as porcine testicular carbonyl reductase, metabolizes androgens and progestins. The enzyme was first isolated from neonatal pig testes, suggesting that 3α/β,20β-HSD may regulate steroid hormone concentrations in the testes during development [1], [2], [3]. Later studies revealed that porcine 3α/β,20β-HSD is expressed in many other tissues including the kidney, liver, brain, lung, thymus, heart, spleen, and adrenal gland, and belongs to the short-chain dehydrogenase/reductase (SDR) superfamily [4], [5]. The wide distribution of 3α/β,20β-HSD suggested a broader role for the enzyme in steroid hormone metabolism. Interestingly, porcine 3α/β,20β-HSD shares a high degree of amino acid homology with human, rat, mouse, and rabbit monomeric carbonyl reductases (CRs) [6], [7], [8], [9]. However, porcine 3α/β,20β-HSD contains 12 amino acids at its carboxyl-terminus that are absent in other mammalian monomeric carbonyl reductases. Nearly all members of the SDR superfamily possess a triad of amino acids—Tyr, Lys, and Ser—that has been identified as catalytically important [10]. The same catalytic triad is also present in 3α/β,20β-HSD and the Tyr and Lys side chains are involved in the catalytic reaction [11]. On the other hand, a Cys residue, not conserved in the SDR superfamily but present in both mammalian NAD-dependent 15-hydroxyprostaglandin dehydrogenase and human carbonyl reductase (also known as 15-hydroxyprostaglandin dehydrogenase/9-ketoprostaglandin reductase) reportedly influences the enzyme activity and reacts with organomercurials [12], [13]. The sequence homology between 3α/β,20β-HSD and human carbonyl reductase is estimated to be about 85% [4]. The tertiary structure of 3α/β,20β-HSD has been determined recently, revealing structural features of a mammalian carbonyl reductase, the first monomeric member of the SDR superfamily [14]. The structure showed the presence of the Cys 226 side chain in close proximity to the nicotinamide ring of the bound NADP molecule.
In this paper, we report the results of modification of the reactive Cys residues in porcine 3α/β,20β-HSD by 5,5′-dithio-bis(2-nitrobenzoic acid) (DTNB) and 4-(hydroxyl mercury) benzoic acid (4-HMB), and discuss the possible roles of Cys 226 in the action of the enzyme and in the binding of cofactor NADPH.
Section snippets
Chemicals
4-Nitrobenzaldehyde was obtained from Nacalai Tesque Inc. (Kyoto, Japan). Menadione, 5,5′-dithio-bis(2-nitrobenzoic acid) (DTNB), 4-(hydroxyl mercury) benzoic acid, sodium salt (4-HMB) and isopropyl-β-d-(−)thiogalactopyranoside (IPTG) were obtained from Wako Pure Chemical Industries Ltd. (Tokyo, Japan). NADPH, NADP+, NAD+, and various nucleotides were obtained from Sigma–Aldrich Japan (Tokyo, Japan). DE-52 was purchased from Whatman International Ltd. (Maidstone, UK). All other reagents were of
The location of Cys residues on the tertiary structure of 3α/β,20β-HSD
The 3α/β,20β-HSD tertiary structure has a basic SDR fold including a seven-stranded parallel β-sheet (βA to βG) flanked by three parallel helices on each side (αB, αC, αG and αD, αE, αF), except for the 41-residue insertion (four helices, αF′-1 to αF′-4) before αF and the addition of a C-terminal strand βH [14]. All six Cys residues of the enzyme are displayed on the tertiary structure shown in Fig. 1a. Residues Cys 25, Cys 73, and Cys 121 belong to helices αB, αD, and αE, respectively. Cys 149
Acknowledgements
This work was supported in part by a grant from the Ministry of Education, Culture, Sports, Science and Technology of Japan. DG is supported in part by National Institutes of Health, USA, grant GM62794.
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