Cloning and Expression of Carbonyl Reductase from Rat Testis

Abstract

Carbonyl reductase is a cytosolic, monomeric, NADPH-dependent oxidoreductase with broad specificity for carbonyl compounds and a general distribution in human and many animal tissues. A carbonyl-reductase-like enzyme is exclusively expressed in rat reproductive tissues and adrenal glands. To understand the structural and functional relationships between the two enzymes, we cloned and sequenced the cDNA for the enzyme from rat testis and overexpressed the encoded protein in Escherichia coli. Three types of cDNA coding for the same protein but differing in their 5'-untranslated regions were isolated. The encoded protein had the same length as, and showed 86% positional identity with, carbonyl reductase from human placenta and liver. The recombinant enzyme exhibited the same substrate specificity and susceptibility to inhibitors as the enzyme isolated from rat testis, and also closely resembled human carbonyl reductase. Similar to the human enzyme, the recombinant rat enzyme catalyzed its own modification by pyruvate and 2-oxoglutarate, respectively. We conclude that carbonyl reductase from rat testis and human tissues represent species-specific forms of the same enzyme.

Carbonyl Reductases and Pluripotent Hydroxysteroid Dehydrogenases of the Short-chain Dehydrogenase/reductase Superfamily

“…Human carbonyl-reducing enzymes occur in many different tissues, such as liver (Ahmed et al, 1979;Felsted and Bachur, 1980;Hara et al, 1986b), lung (Nakayama et al, 1986;Nakanishi et al, 1995), heart (Imamura et al, 1999a;Imamura et al, 1999b), kidney Imamura et al, 1993), brain (Wermuth, 1981;Cromlish et al, 1985;Iwata et al, 1989;Iwata et al, 1990b;Inazu et al, 1992b;Wermuth et al, 1995), ovary (Iwata et al, 1989;Aoki et al, Drug Metabolism Reviews Downloaded from informahealthcare.com by Michigan University on 11/01/14…”

3α/β,20β-Hydroxysteroid dehydrogenase (porcine testicular carbonyl reductase) also has a cysteine residue that is involved in binding of cofactor NADPH

“…The wide distribution of 3␣/␤,20␤-HSD suggested a broader role for the enzyme in steroid hormone metabolism. Interestingly, porcine 3␣/␤,20␤-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 3␣/␤,20␤-HSD contains 12 amino acids at its carboxyl-terminus that are absent in other mammalian monomeric carbonyl reductases.…”