No abstract

Neuroactive steroids are naturally occurring metabolites of endogenous steroid hormones, which exert rapid and nongenomic effects on membrane-bound neurotransmitter receptors. Those synthesized in the brain, termed neurosteroids, are believed to alter neuronal excitability through interaction with specific neurotransmitter receptors.1) Of the neurosteroids, 3a,5a-tetrahydroprogesterone (3a,5a-THP) and 3a,5a-tetrahydrodeoxycorticosterone (3a,5a-THDOC) are the most potent and efficacious of known positive allosteric modulators of g-aminobutyric acid type A (GABA A ) receptors.1-3) Numerous animal studies have shown that 3a,5a-THP and 3a,5a-THDOC are synthesized from progesterone and deoxycorticosterone, respectively, through the respective intermediates, 5a-dihydroprogesterone (5a-DHP) and 5a-dihydrodeoxycorticosterone (5a-DHDOC).3,4) The two sequential enzymatic reactions are catalyzed by steroid 5a-reductase and cytosolic NADPH-dependent 3a-hydroxysteroid dehydrogenase (3a-HSD, EC 1.1.1.213). On the other hand, microsomal NAD ϩ -dependent 3a-HSD oxidizes the neurosteroids back to 5a-DHP and 5a-DHDOC, and is thought to be involved in the catabolism of the potent GABA A ergic steroids.In the human, four isoenzymes of cytosolic NADP(H)-dependent 3a-HSD, which share at least 83% amino acid sequence identity and belong to the aldo-keto reductase (AKR) family, 5) have been identified. According to the nomenclature for the AKR family, these are termed AKR1C1, AKR1C2, AKR1C3 and AKR1C4, which correspond to previously known 3(20)a-HSD or 20a-HSD, 6,7) type 3 3a-HSD, 8) type 2 3a-HSD 9,10) and type 1 3a-HSD, 10) respectively. The four isoenzymes have recently been demonstrated to exhibit broad substrate specificities for 3a-, 17b-and 20a-hydroxysteroids, 11) but the preferences for the respective types of steroid substrates are different among the isoenzymes, as AKR1C1 shows high 20a-HSD activity 6) and AKR1C3 has been shown to be identical to type 5 17b-HSD 12) and prostaglandin F synthase. 13,14) Analyses of mRNA species for AKR1C isoenzymes in human tissues have shown that the isoenzymes, except for liver-specific AKR1C4, are expressed ubiquitously. 7,[9][10][11][14][15][16][17] In human brain AKR1C1, AKR1C2 and AKR1C3 are highly expressed, 11,16) although the distribution and localization of the enzymes in the brain remain unknown. These findings have suggested not only the roles of AKR1C1-AKR1C3 in the synthesis of the 3a,5a-THP and 3a,5a-THDOC, but also a possibility that the isoenzymes convert the potent neurosteroids to weak neurosteroids, 3a,20a-dihydroxypregnanes, 2,3) by exhibiting their 20a-HSD activities. However, there is limited information on the substrate specificity of human AKR1C isoenzymes for the neurosteroids and their precursors. To address the roles of human AKR1C isoenzymes in the metabolism of the neurosteroids, kinetic constants for the steroids have been determined with homogeneous recombinant AKR1C1-AKR1C3. Furthermore, we have found that several benzodiazepines, which bind to the GABA A recep...

Japanese monkey liver contains multiple forms of dihydrodiol dehydrogenase with 3(20)alpha-hydroxysteroid dehydrogenase activity. Here we have purified the major and minor forms (DD1 and DD4) of the enzyme from Cynomolgus monkey liver, and isolated cDNA species for the two enzyme forms by reverse transcription-PCR. The cDNAs encoded proteins comprising of 323 amino acids, in which the sequence identity between DD1 and DD4 was 83%. The sequences deduced from the cDNAs for DD1 and DD4 perfectly matched the partial sequences of peptides derived from the respective enzymes. We also isolated the cDNAs for DD1 and DD4 of Japanese monkey liver, which had almost identical amino acid sequences with those of the respective enzymes of Cynomolgus monkey liver. The monkey DD1s and DD4s showed the highest sequence identity (94%) with AKR1C1 and AKR1C4, respectively, of four isoenzymes of human 3(20)alpha-hydroxysteroid dehydrogenase, which belongs to the aldo-keto reductase family. The substrate specificity and inhibitor sensitivity of the purified recombinant Cynomolgu monkey DD1 and Japanese monkey DD4 were also essentially identical to those of the recombinant AKR1C1 and AKR1C4, respectively, indicating that DD1 and DD4 are homologues of human AKR1C1 and AKR1C4, respectively. The mRNA for DD1 was detected only in liver, kidney, intestine and adrenal gland among Japanese monkey tissues, and that for DD4 was expressed in liver and kidney. These tissue distribution patterns differ from those of human AKR1C1 and AKR1C4, which are expressed ubiquitously and liver-specific, respectively. In addition, no mRNA for an enzyme corresponding to another isoenzyme (AKR1C2) of the human enzyme was detected in livers of the two monkey strains. The results suggest a difference in the metabolism of steroids and xenobiotics mediated by 3(20)alpha-hydroxysteroid dehydrogenase isoenzymes between monkeys and humans.

The NADP(H)-linked oxidoreductase activity of a major isozyme of human liver 3 alpha-hydroxysteroid dehydrogenase was activated 5-, 4-, and 2-fold by D-thyroxine (T(4)), L-T(4) and DL-3,3', 5'-triiodothyronine (reverse T(3)), respectively. Kinetic analysis of the activation indicated that D-T(4), L-T(4), and reverse T(3) are non-essential activators, showing binding constants of 1.5, 1.1, and 3.6 microM, respectively. Comparison of the effects of the T(4) analogs on the activities of the mutant enzymes suggests that the binding site is composed of at least Lys-270, Arg-276, and the C-terminal loop of the enzyme. L-T(3), DL-thyronine, and D-tyrosine had no effect on the enzyme, but 3,5,3',5'-tetra- and 3,5, 3'-tri-iodo thyropropionic acids were potent competitive inhibitors with K(i) values of 42 and 60 nM, respectively, with respect to the substrate. The inhibition constant was lowered upon the activation of the enzyme by D-T(4), and the inhibition by the deamino derivatives of T(4) and T(3) disappeared upon modification of the C-terminal loop of the enzyme, but not upon replacement of Lys-270 or Arg-276 with Met. These results indicate that, depending on their structures, the T(4) analogs bind differently to two distinct sites at the active center of the enzyme to produce stimulatory and inhibitory effects.

Human dihydrodiol dehydrogenase with 3alpha-hydroxysteroid dehydrogenase activity exists in four forms (AKR1C1-1C4) that belong to the aldo-keto reductase (AKR) family. Recent crystallographic studies on the other proteins in this family have indicated a role for a tyrosine residue (corresponding to position 216 in these isoenzymes) in stacking the nicotinamide ring of the coenzyme. This tyrosine residue is conserved in most AKR family members including AKR1C1-1C3, but is replaced with histidine in AKR1C4 and phenylalanine in some AKR members. In the present study we prepared mutant enzymes of AKR1C4 in which His-216 was replaced with tyrosine or phenylalanine. The two mutations decreased 3-fold the K(m) for NADP(+) and differently influenced the K(m) and k(cat) for substrates depending on their structures. The kinetic constants for bile acids with a 12alpha-hydroxy group were decreased 1.5-7-fold and those for the other substrates were increased 1.3-9-fold. The mutation also yielded different changes in sensitivity to competitive inhibitors such as hexoestrol analogues, 17beta-oestradiol, phenolphthalein and flufenamic acid and 3,5,3', 5'-tetraiodothyropropionic acid analogues. Furthermore, the mutation decreased the stimulatory effects of the enzyme activity by sulphobromophthalein, clofibric acid and thyroxine, which increased the K(m) for the coenzyme and substrate of the mutant enzymes more highly than those of the wild-type enzyme. These results indicate the importance of this histidine residue in creating the cavity of the substrate-binding site of AKR1C4 through the orientation of the nicotinamide ring of the coenzyme, as well as its involvement in the conformational change by binding non-essential activators.