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 carbonylreductase-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.

Tumor-related protein, the squamous cell carcinoma antigen binds to the intracellular protein carbonyl reductase

“…CR is a NADPH-dependent, mostly monometric, cytosolic enzyme with broad substrate specificity for carbonyl compounds (17,18). It has been therefore extensively studied in relation to its ability to reduce a variety of carbonyl conpounds; the antitumor anthracycline antibiotics, daunorubicin (19) and doxorubicin, and prostaglandins (20).…”

Cloning and bacterial expression of monomeric short-chain dehydrogenase/reductase (carbonyl reductase) from CHO-K1 cells

“…The primers (CRf01 and CRr01) for the first PCR were designed from the conserved nucleotide sequences of mammalian CR cDNAs as shown in Fig. 1 (human CR, human carbonyl reductase; pig 20bHSD, pig 20b-hydroxysteroid dehydrogenase; rat CR, rat carbonyl reductase; mouse CR, mouse carbonyl reductase 2) [5,16,37,38].…”

“…r, y, s and w indicate A or G, C or T, C or G and A or T, respectively. [16]; pig 20bHSD, pig 20b-hydroxysteroid dehydrogenase [5]; rat CR, rat carbonyl reductase [37]; mouse CR, mouse carbonyl reductase 2 [38].`r',`y',`s' and`w' indicate`A or G',`C or T',`C or G' and`A or T', respectively. 0.2 mm IPTG was added to the medium and incubated for a further 3.5 h. Bacterial cells were then harvested and washed with 20 mm NaCl/P i , pH 7.5 (buffer A).…”

“…Homology scores of carbonyl reductase in nucleotide and amino-acid sequences. AA, amino acid; NN, nucleotide; CHO-CR, this study; human CBR3 [40]; human CR, human carbonyl reductase [16]; pig 20b-HSD, pig 20b-hydroxysteroid dehydrogenase [5]; rabbit CR, rabbit carbonyl reductase [45]; rat CR, rat testis carbonyl reductase [37]; mouse CR2, mouse carbonyl reductase 2 [38].…”

“…5). RT-PCR was carried out using the primers ; hCR, human carbonyl reductase [16]; pig 20bHSD, pig 20b-hydroxysteroid dehydrogenase [5]; rab CR, rabbit carbonyl reductase [46]; rat CR, rat carbonyl reductase [37]; mouse CR, mouse carbonyl reductase 2 [38]. Open squares indicate NADP 1 -binding (TGxxxGxG) and SDR family active site motifs (S-Y-K), respectively.…”