Human 3alpha-hydroxysteroid dehydrogenase exists in four isoforms, which belong to the aldo-keto reductase (AKR) superfamily and are named AKR1C1-AKR1C4. The properties of AKR1C3 have not been fully characterized compared to the other isoforms. In addition, a cDNA that shows more than 99% homology with AKR1C3 cDNA has been cloned from human myeloblasts. We have here expressed and purified a recombinant enzyme (designated as DBDH) from this cDNA. DBDH oxidized xenobiotic alicyclic alcohols and 3alpha- or 17beta-hydroxy-5beta-androstanes, and catalyzed the reversible conversion between prostaglandin D2 and 9alpha,11beta-prostaglandin F2 more efficiently than that of 3alpha- or 17beta-hydroxysteroids:the respective Km values were 0.6 and 6.8 microM, and kcat/Km values were about 1,000 min-1.mM-1. Anti-inflammatory drugs highly inhibited the enzyme. The recombinant AKR1C3 prepared by site-directed mutagenesis of DBDH also showed the same properties as the wild-type DBDH. Analyses of expression of mRNAs for DBDH and AKR1C3 by reverse transcription-PCR indicated that only one mRNA species for DBDH is expressed in 33 human specimens of liver, kidney, lung, brain, heart, spleen, adrenal gland, small intestine, placenta, prostate, and testis. These results suggest that AKR1C3 acts as prostaglandin D2 11-ketoreductase, and that its principal gene in the human has a coding region represented by DBDH cDNA.

We previously isolated three monomeric dihydrodiol dehydrogenases, DD1, DD2 and DD4, from human liver, and cloned a cDNA (C9) thought to encode DD2, which is identical with those for human bile-acid-binding protein and an oxidoreductase of human colon carcinoma HT29 cells. In the present study we have provided evidence that the C9 cDNA clone encodes DD1, not DD2. A recombinant enzyme expressed from the cDNA in a bacterial system was purified, and its catalytic properties, bile-acid-binding ability and primary sequence were compared with those of the hepatic dihydrodiol dehydrogenases. The results show that DD1 encoded by C9 possesses prostaglandin F synthase activity but low affinity for lithocholic acid, whereas DD2, showing differences of six amino acid residues from the DD1 sequence, exhibited high-affinity binding for the bile acid. Refined relationship between dihydrodiol dehydrogenases and their related proteins of human tissues is proposed.

Several mammalian livers contain monomeric 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD) with A-stereospecificity in hydrogen transfer, which differs from the B-specific dimeric enzyme of human placenta in its ability to catalyze the oxidoreduction of xenobiotic trans-dihydrodiols of aromatic hydrocarbons and carbonyl compounds. Here, we report the isolation and characterization of a mouse cDNA clone encoding monomeric 17 beta-HSD of the liver. This clone had an entire coding region for a protein of 323 amino acid residues with a molecular weight of 37,055. The deduced sequence of the protein aligned with a high degree of identity with rat and rabbit 20 alpha-HSDs, rat and human 3 alpha-HSD/dihydrodiol dehydrogenases, and bovine prostaglandin F synthase, which are members of the aldoketoreductase family, but was distinct from human 17 beta-HSD and carbonyl reductase, members of the short chain dehydrogenases. The expression of the cDNA in Escherichia coli resulted in synthesis of a protein that was active toward androgens, estrogens, and xenobiotic substrates. The recombinant and mouse liver 17 beta-HSDs also exhibited low 20 alpha-HSD activity toward progestins, which is similar to bifunctional activity of human placental 17 beta-HSD. Therefore, the mouse enzyme was given the designation of estradiol 17 beta-dehydrogenase (A-specific). Northern analysis of mouse tissues revealed the existence of a single 1.7-kilobase 17 beta-HSD mRNA species in the liver, kidney, testis, and stomach. The liver mRNA content was considerably more abundant than those found in the other tissues, as 17 beta-HSD protein was mainly detected in the liver by Western analysis.

Human liver contains two dihydrodiol dehydrogenases, DD2 and DD4, associated with 3 alpha-hydroxysteroid dehydrogenase activity. We have raised polyclonal antibodies that cross-reacted with the two enzymes and isolated two 1.2 kb cDNA clones (C9 and C11) for the two enzymes from a human liver cDNA library using the antibodies. The clones of C9 and C11 contained coding sequences corresponding to 306 and 321 amino acid residues respectively, but lacked 5'-coding regions around the initiation codon. Sequence analyses of several peptides obtained by enzymic and chemical cleavages of the two purified enzymes verified that the C9 and C11 clones encoded DD2 and DD4 respectively, and further indicated that the sequence of DD2 had at least additional 16 residues upward from the N-terminal sequence deduced from the cDNA. There was 82% amino acid sequence identity between the two enzymes, indicating that the enzymes are genetic isoenzymes. A computer-based comparison of the cDNAs of the isoenzymes with the DNA sequence database revealed that the nucleotide and amino acid sequences of DD2 and DD4 are virtually identical with those of human bile-acid binder and human chlordecone reductase cDNAs respectively.

Functionalization of endohedral metallofullerenes has been shown to differ depending on photochemical or thermal pathways. We report that Lu(3)N@I(h)-C(80) reacts with thermally generated bis(2,6-diethylphenyl)silylene with high selectivity and forms monosilylated derivative 1b. Unexpectedly, 1b undergoes photochemical conversion to afford isomer 1a under ambient light. These adducts were characterized using NMR, visible-near-IR spectroscopy, and matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Single-crystal X-ray structure determination of 1a reveals a rare example of an open 1,2-adduct at the [5,6]-ring junction of the I(h)-C(80) cage. The electrochemical study reveals that the redox potentials of 1a and 1b are shifted cathodically compared to those of pristine Lu(3)N@I(h)-C(80) and that monosilylation is effective to fine-tune the electronic properties of endohedral metallofullerenes as well as empty fullerenes. Density functional theory calculations were also performed, which provide a theoretical basis for the structures and the behavior of the encapsulated Lu(3)N cluster.