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Akira Hara - One of the best experts on this subject based on the ideXlab platform.
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Dimeric Dihydrodiol Dehydrogenase is an efficient primate 1,5-anhydro-D-fructose reductase
Biochemical and biophysical research communications, 2020Co-Authors: Akira Hara, Ossama El-kabbani, Toru Nishinaka, Naohito Abe, Toshiyuki Matsunaga, Satoshi EndoAbstract:Abstract 1,5-Anhydro-D-fructose (AF), a metabolite of the anhydrofructose pathway of glycogen metabolism, has recently been shown to react with intracellular proteins and form advanced glycation end-products. The reactive AF is metabolized to non-reactive 1,5-anhydro-D-glucitol by AF reductase in animal tissues and human cells. Pig and mouse AF reductases were characterized, but primate AF reductase remains unknown. Here, we examined the AF-reducing activity of eleven primate NADPH-dependent reductases with broad substrate specificity for carbonyl compounds. AF was reduced by monkey dimeric Dihydrodiol Dehydrogenase (DHDH), human aldehyde reductase (AKR1A1) and human dicarbonyl/L-xylulose reductase (DCXR). DHDH showed the lowest KM (21 μM) for AF, and its kcat/KM value (1208 s−1mM−1) was much higher than those of AKR1A1 (1.3 s−1mM−1), DCXR (1.1 s−1mM−1) and the pig and mouse AF reductases. AF is a novel substrate with higher affinity and catalytic efficiency than known substrates of DHDH. Docking simulation study suggested that Lys156 in the substrate-binding site of DHDH contributes to the high affinity for AF. Gene database searches identified DHDH homologues (with >95% amino acid sequence identity) in humans and apes. Thus, DHDH acts as an efficient AF reductase in primates.
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Structure of monkey dimeric Dihydrodiol Dehydrogenase in complex with isoascorbic acid.
Acta crystallographica. Section D Biological crystallography, 2008Co-Authors: Vincenzo Carbone, Akira Hara, Rie Sumii, Shuhei Ishikura, Yukuhiko Asada, Ossama El-kabbaniAbstract:Mammalian dimeric Dihydrodiol Dehydrogenase (DD) is identical to NADP+-dependent D-xylose Dehydrogenase. A recent investigation showed that the three-dimensional structure of monkey DD is similar to those of prokaryotic NADP(H)-dependent glucose-fructose oxidoreductase (GFO) and 1,5-anhydro-D-fructose reductase (AFR); however, it differs in coenzyme-binding and catalytic residues. Dimeric DD has a high affinity for NADP(H) when compared with AFR and differs from both GFO and AFR in its specificity for sugars and hydrophobic xenobiotic compounds as substrates. The crystal structure of monkey dimeric DD complexed with the inhibitor isoascorbic acid has been determined at 2.59 angstroms resolution. Molecular modelling of coenzyme binding complemented with site-directed mutagenesis has been utilized to propose a binding mode for the coenzyme molecule and to gain insights into the roles of the residues comprising the active site and coenzyme-binding domain of DD. Several key residues have been identified within the coenzyme-binding domain, including Arg37, Arg41, His76 and His79, that contribute to the high affinity for coenzyme. The interaction of Arg37 and Arg41 with the 2'-phosphate and adenine-ring moiety of the coenzyme has been established from the large increases (29-fold to 438-fold) in the Kd values for NADP(H) for the R37D and R41D mutant enzymes. The mutation of several residues lining the inhibitor-binding site of DD suggested the involvement of Trp125, Phe154, Trp254 and Phe279 in determining the broad substrate specificity and inhibitor potency of the enzyme. In addition, mutants of Lys97, which is present near the catalytic residue Tyr180, greatly reduced the kcat value without changing the Kd values for coenzyme, suggesting the importance of Lys97 in the catalytic mechanism of DD.
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structures of dimeric Dihydrodiol Dehydrogenase apoenzyme and inhibitor complex probing the subunit interface with site directed mutagenesis
Proteins, 2007Co-Authors: Vincenzo Carbone, Akira Hara, Rie Sumii, Roland P.-t. Chung, Toshiyuki Matsunaga, Satoshi Endo, Ossama ElkabbaniAbstract:Dimeric Dihydrodiol Dehydrogenase (DD) catalyses the nicotinamide adenine dinucleotide phosphate (NADP+)-dependent oxidation of trans-Dihydrodiols of aromatic hydrocarbons to their corresponding catechols. This is the first report of the crystal structure of the dimeric enzyme determined at 2.0 A resolution. The tertiary structure is formed by a classical dinucleotide binding fold comprising of two betaalphabetaalphabeta motifs at the N-terminus and an eight-stranded, predominantly antiparallel beta-sheet at the C-terminus. The active-site of DD, occupied either by a glycerol molecule or the inhibitor 4-hydroxyacetophenone, is located in the C-terminal domain of the protein and maintained by a number of residues including Lys97, Trp125, Phe154, Leu158, Val161, Asp176, Leu177, Tyr180, Trp254, Phe279, and Asp280. The dimer interface is stabilized by a large number of intermolecular contacts mediated by the beta-sheet of each monomer, which includes an intricate hydrogen bonding network maintained in principal by Arg148 and Arg202. Site-directed mutagenesis has demonstrated that the intact dimer is not essential for catalytic activity. The similarity between the quaternary structures of mammalian DD and glucose-fructose oxidoreductase isolated from the prokaryotic organism Zymomonas mobilis suggests that both enzymes are members of a unique family of oligomeric proteins and may share a common ancestral gene.
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Crystallization and preliminary X-ray diffraction analysis of monkey dimeric Dihydrodiol Dehydrogenase
Acta Crystallographica Section D Biological Crystallography, 2001Co-Authors: Ossama El-kabbani, Yukuhiko Asada, Roland P.-t. Chung, Paul A. Ramsland, Syuhei Ishikura, Noriyuki Usami, Akira HaraAbstract:Dihydrodiol Dehydrogenase catalyzes the NADP+-linked oxidation of trans-Dihydrodiols of aromatic hydrocarbons to corresponding catechols and exists in multiple forms in mammalian tissues. The dimeric form of mammalian Dihydrodiol Dehydrogenase has a primary structure distinct from the previously known mammalian enzymes and may constitute a novel protein family with the prokaryotic proteins. Monkey kidney dimeric Dihydrodiol Dehydrogenase was crystallized from buffered ammonium phosphate solution using the hanging-drop vapour-diffusion method. The crystals diffract to 2.65 A resolution in the laboratory and belong to the hexagonal P6122 or P6522 space group, with unit-cell parameters a = b = 122.8, c = 121.3 A, α = β = 90, γ = 120°.
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Crystallization and preliminary X-ray diffraction analysis of monkey dimeric Dihydrodiol Dehydrogenase.
Acta crystallographica. Section D Biological crystallography, 2001Co-Authors: Ossama El-kabbani, Yukuhiko Asada, Roland P.-t. Chung, Paul A. Ramsland, Syuhei Ishikura, Noriyuki Usami, Akira HaraAbstract:Dihydrodiol Dehydrogenase catalyzes the NADP(+)-linked oxidation of trans-Dihydrodiols of aromatic hydrocarbons to corresponding catechols and exists in multiple forms in mammalian tissues. The dimeric form of mammalian Dihydrodiol Dehydrogenase has a primary structure distinct from the previously known mammalian enzymes and may constitute a novel protein family with the prokaryotic proteins. Monkey kidney dimeric Dihydrodiol Dehydrogenase was crystallized from buffered ammonium phosphate solution using the hanging-drop vapour-diffusion method. The crystals diffract to 2.65 A resolution in the laboratory and belong to the hexagonal P6(1)22 or P6(5)22 space group, with unit-cell parameters a = b = 122.8, c = 121.3 A, alpha = beta = 90, gamma = 120 degrees.
Kuan-chih Chow - One of the best experts on this subject based on the ideXlab platform.
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expression of Dihydrodiol Dehydrogenase plays important roles in apoptosis and drug resistance of a431 squamous cell carcinoma
Journal of Dermatological Science, 2006Co-Authors: Kuan-chih ChowAbstract:Summary Background Dihydrodiol Dehydrogenase (DDH) is a member of the aldo-keto reductases superfamily which may be involved in normal detoxification process of environmental mutagenic hazards like polycyclic aromatic hydrocarbons (PAH). Previous clinical studies have demonstrated the over-expression of DDH in various types of cancers, including cutaneous squamous cell carcinoma (SCC), and its correlation with tumor progression and grave prognosis. Objective To investigate possible mechanisms for DDH's correlation with tumor progression and unfavorable prognosis. Methods DDH expression in SCC A431 cell line was examined by quantitative real-time PCR and immunoblotting. RNA interference (RNAi) by transduction with retroviral vector containing DDH-targeting small interfering RNA was employed to inhibit DDH expression by A431 cells. With DDH expression inhibited or not, sensitivity of A431 cells to UVB-induced apoptosis and cytotoxicity of chemotherapeutic agent bleomycin were then examined and compared. Results DDH was found highly expressed by SCC A431 cells, which was barely detectable in other normal or malignant cutaneous cells, including keratinocytes, fibroblast, and basal cell carcinoma cell line. RNAi Inhibition of DDH expression in A431 cells led to increased sensitivity to UVB-induced apoptosis and cytotoxicity of bleomycin treatment. Conclusion DDH may play important roles in tumor progression of SCC via induction of apoptosis- and drug-resistance.
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Infection of human papillomavirus and overexpression of Dihydrodiol Dehydrogenase in uterine cervical cancer.
Gynecologic oncology, 2006Co-Authors: Masatsugu Ueda, Shiow-her Chiou, Tze-yi Lin, Yao Ching Hung, Jin Tang Chen, Hsuan Hua Huang, Yoshito Terai, Kuan-chih ChowAbstract:Abstract Objective To determine the association of human papillomavirus (HPV) infection with the expression of Dihydrodiol Dehydrogenase (DDH) in uterine cervical cancer (UCC). Methods In situ hybridization (ISH) and immunohistochemistry were applied to examine pathological specimens of 145 patients with UCC. Results By ISH, HPV16/18 DNA was detected in 108 (74.5%) UCC cases. DDH expression determined by immunohistochemistry was detected in 81 (75%) lesions among 108 HPV-positive cases. In contrast, of 37 HPV-negative cases, DDH was only detected in 16 (43.2%) of the lesions. A significant correlation was found between DDH expression and the presence of HPV ( P P = 0.004), lymph node involvement ( P P = 0.002). In vitro, DDH expression was also found closely associated with HPV infection, and DDH content was proportional to cell sensitivity for cisplatin and doxorubicin. Conclusions HPV infection provokes local inflammation, which can then induce DDH expression and drug resistance in UCC. The detailed biological relationship among HPV infection, expression of DDH and drug resistance, however, remains to be clarified.
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Expression of Dihydrodiol Dehydrogenase and Resistance to Chemotherapy and Radiotherapy in Adenocarcinoma Cells of Lung
Anticancer research, 2006Co-Authors: Jung-jyh Hung, Kuan-chih Chow, Hao-wei Wang, Liang-shun WangAbstract:Background: The cytoplasmic enzyme Dihydrodiol Dehydrogenase (DDH) plays an important role in the detoxification process. Recently, the overexpression of DDH was detected in non-small cell lung cancer (NSCLC) cells and patients with DDH overexpression were shown to have a significantly higher incidence of early tumor recurrence and distant metastasis. In this study, the correlation between DDH expression and resistance to cisplatin, adriamycin and radiotherapy in NSCLC was examined. Materials and Methods: Seven lung adenocarcinoma cell lines (H23, H838, H1437, H1648, H2009, H2087 and H2126) were used in the study. The DDH level was determined by reverse transcription polymerase chain reaction and immunoblotting. Drug- and radiation-mediated cytotoxicity was measured by clonogenic assay. DDH isoforms (DDH1, DDH2 and DDH3) were transfected into H23 cells that did not express DDH to examine their effects on drug and radiation resistance. Results: DDH-overexpressed adenocarcinoma cells exhibited a much higher resistance to doxorubicin, cisplatin and irradiation than cells with lower DDH expression. The DDH2- and DDH1-transfectants showed higher drug and radiation resistance than the DDH3-trasnfectant. Conclusion: Resistance to both anticancer drugs and irradiation in lung adenocarcinoma cells was closely associated with DDH activity. DDH1 and DDH2 were the main isoforms responsible for these effects. Lung cancer is one of the leading causes of cancer death
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Reduction of Dihydrodiol Dehydrogenase expression in resected hepatocellular carcinoma
Oncology reports, 2003Co-Authors: Mei Due Yang, Shiow-her Chiou, Chang-fang Chiu, Tze-yi Lin, I-ping Chiang, Kuan-chih ChowAbstract:Dihydrodiol Dehydrogenase (DDH) is one of the major enzymes catabolizing polycyclic aromatic hydrocarbons in the liver. Although four DDH isoforms have been detected in the normal liver, only DDH1 and DDH2 have been detected in cancer cells of lung and esophagus. Moreover, the available information about hepatic pathophysiological regulation of DDH expression is limited. Therefore we addressed the question of DDH expression in patients with liver disorders, in particular, patients with hepatocellular carcinoma (HCC). Expression of DDH1/2 was determined by immunohistochemistry, immunoblotting and reverse transcription-polymerase chain reaction (RT-PCR) in 52 patients with resected HCC. DDH1/2 expression was detected in 31 (59.6%) of 52 pathological sections. Frequency of DDH1/2 expression was significantly higher in patients with tumor size >2 cm, and in those who had early local recurrence. In addition to the tumor size and frequency of local recurrence, our results further indicated that expression of DDH1/2 was correlated with those of cyclooxygenase 2 (COX-2), interleukin-6 (IL-6), microsomal epoxide hydrolase (mEpH) and soluble epoxide hydrolase (sEpH) in HCC patients. Interestingly, the expression of DDH1/2 was found inversely correlated with that of glutathione S-transferase (GST) and NADPH p450 reductase (NPR). In conclusion, these results indicate that DDH expression was significantly decreased in about 40% of HCC patients. However, in the bordering non-neoplastic region of liver DDH1/2 expression increased, and the increased DDH1/2 expression correlated with tumor size and the disease progression.
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expression of Dihydrodiol Dehydrogenase in the resected stage i non small cell lung cancer
Oncology Reports, 2002Co-Authors: Chih Yi Chen, Tze-yi Lin, Chung Ping Hsu, Nan Yung Hsu, Chih Shiun Shih, Kuan-chih ChowAbstract:Recently, by using differential display on specimens of non-small cell lung cancer (NSCLC), we detected overexpression of Dihydrodiol Dehydrogenase (DDH) that was rarely expressed in the corresponding normal lung tissue. DDH overexpression was correlated with poor prognosis of patients with advanced NSCLC. Because DDH could metabolize polycyclic aromatic hydrocarbons (PAH) in the liver, DDH overexpression in NSCLC would suggest an association with carcinogenesis and disease progression. In this study, we investigated DDH expression in 103 patients with resected stage I NSCLC. Expression of DDH was detected by using immunohistochemistry. Relation between DDH expression and clinicopathological parameter (age, gender, smoking habit, tumor status, histological type, cell differentiation, local recurrence, distant metastasis or survival) was analyzed by statistical analysis. DDH overexpression was detected in 39 (37.9%) of 103 pathological sections. Frequency of DDH overexpression was significantly higher in male patients (p=0.043) and patients with squamous cell carcinoma (p<0.005). Among 103 patients, 14 patients had local recurrence and 28 patients had distant metastasis during follow-up examination. The 5-year survival rate of these patients was poorer than those who did not have local recurrence or distant metastasis (both were p<0.005, respectively). Although patients with low DDH expression had more favorable outcome than those with DDH overexpression, in terms of survival rate no statistical significance was detected (p=0.889). The results suggest that DDH expression may serve as an early but not prognostic biomarker for patients with resectable stage I NSCLC.
Trevor M. Penning - One of the best experts on this subject based on the ideXlab platform.
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Stable expression of rat Dihydrodiol Dehydrogenase (AKR1C9) in human breast MCF-7 cells results in the formation of PAH-o-quinones and enzyme mediated cell death.
Chemical research in toxicology, 2001Co-Authors: Laurie S. Tsuruda, Trevor M. PenningAbstract:Dihydrodiol Dehydrogenase members of the aldo-keto reductase (AKR) superfamily have been implicated in the metabolic activation of PAH trans-Dihydrodiols because they convert these proximate carcinogens to reactive and redox-active o-quinones. In this study, rat liver 3alpha-hydroxysteroid/Dihydrodiol Dehydrogenase (AKR1C9) was stably transfected into human breast carcinoma (MCF-7) cells, which represent a null-environment for AKR expression, to detect the formation of PAH o-quinones in a cellular context and the cellular consequences of o-quinone formation. The heterologous transfected cells expressed AKR1C9 mRNA and protein. Immunotitration of the enzyme activity indicated that the expressed protein constituted 1.0% of the soluble protein. The specific activity of the expressed enzyme was also comparable to that observed in rat liver cytosol. The transfectants were found to convert (+/-)-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene (B[a]P-7,8-diol) to benzo[a]pyrene-7,8-dione (BPQ). The identity of this metabolite was confirmed by co-chromatography and by UV-Vis diode-array spectrometry. This conversion was not evident in mock-transfected cells. The cytotoxic consequences of BPQ formation was also examined. Transfectants exposed to 1 microM B[a]P-7,8-diol revealed that cytotoxicity, as measured by lactate Dehydrogenase (LDH) release, occurred over the time course of o-quinone formation leading to 77% of the cellular LDH being released by 16 h. AKR1C9 inhibitors blocked the B[a]P-7,8-diol dependent cytotoxicity indicating that it was mediated by the enzymatically formed BPQ. These data indicate that high stable constitutive expression of AKR1C9 will result in B[a]P-7,8-diol mediated cytotoxicity due to the formation of unconjugated BPQ.
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Polycyclic Aromatic Hydrocarbon Trans-Dihydrodiol Specificity of four Recombinant Human Dihydrodiol Dehydrogenase Isoforms
Polycyclic Aromatic Compounds, 2000Co-Authors: Michael E. Burczynski, Ronald G. Harvey, Nisha T. Palackal, Trevor M. PenningAbstract:Abstract A major metabolic route of polycyclic aromatic hydrocarbon (PAH) activation proceeds through trans-Dihydrodiol intermediates. We have previously shown that a member of the aldo-keto reductase (AKR) superfamily, rat liver Dihydrodiol Dehydrogenase (DD), catalyzes the NAD(P)+-dependent oxidation of PAH trans-Dihydrodiols with the concomitant production of reactive oxygen species and o-semiquinone anion radicals on route to cyto- and geno-toxic o-quinones. The relevance of this pathway in humans, however, is unknown. In these studies, four homogeneous recombinant human homologs of rat liver DD (DD1, DD2, DD4 and DDX) were tested for their ability to oxidize a structural series of PAH trans-Dihydrodiols of increasing ring size and methylation. The results indicate that human DDs preferred non-K-region trans-Dihydrodiols and that methyl substitution enhanced oxidation rates by human DDs. Thus multiple human AKRs can contribute to the activation of structurally diverse procarcinogenic PAH by catalyzing...
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Genomic structure of rat 3α-hydroxysteroid/Dihydrodiol Dehydrogenase (3α-HSD/DD, AKR1C9)
The Journal of Steroid Biochemistry and Molecular Biology, 1999Co-Authors: Hsueh Kung Lin, Chien Fu Hung, Margaret Moore, Trevor M. PenningAbstract:Rat liver 3alpha-hydroxysteroid/Dihydrodiol Dehydrogenase (3alpha-HSD/DD) is a member of the aldo-keto reductase (AKR) superfamily. It is involved in the inactivation of steroid hormones and the metabolic activation of polycyclic aromatic hydrocarbons (PAH) by converting trans-Dihydrodiols into reactive and redox-active o-quinones. The structure of the 5'-flanking region of the gene and factors involved in the constitutive and regulated expression of this gene have been reported [H.-K. Lin, T.M. Penning, Cloning, sequencing, and functional analysis of the 5'-flanking region of the rat 3alpha-hydroxysteroid/Dihydrodiol Dehydrogenase gene, Cancer Res. 55 (1995) 4105-4113]. We now describe the complete genomic structure of the rat type 1 3alpha-HSD/DD gene. Charon 4A and P1 genomic clones contained at least three rat genes (type 1, type 2 and type 3 3alpha-HSD/DD) each of which encoded for the same open reading frame (ORF) but differed in their exon-intron organization. 5'-RACE confirmed that the type 1 3alpha-HSD/DD gene encodes for the dominant transcript in rat liver and it was the regulation of this gene that was previously studied. The rat type 1 3alpha-HSD/DD gene is 30 kb in length and consists of nine exons and eight introns. Exon 9 encodes +931 to 966 bp of the ORF and the 1292 bp 3'-UTR implicated in mRNA stability. This genomic structure is nearly identical to the homologous human genes, type 1 3alpha-HSD (chlordecone reductase/DD4, AKR1C4), type 2 3alpha-HSD (AKR1C3) and type 3 3alpha-HSD (bile-acid binding protein, AKR1C2) genes. Three different cDNA's containing identical ORFs for 3alpha-HSD have been reported suggesting that all three genes may be expressed in rat liver. Using 5' primers corresponding to the 5'-UTR's of the three different cDNA's only one PCR fragment was obtained and corresponded to the type 1 3alpha-HSD/DD gene. These data suggested that the type 2 and type 3 3alpha-HSD/DD genes are not abundantly expressed in rat liver. It is unknown whether the type 2 and type 3 3alpha-HSD/DD genes represent pseudo-genes or whether they represent genes that are differentially expressed in other rat tissues.
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Dexamethasone Regulation of the Rat 3α-Hydroxysteroid/Dihydrodiol Dehydrogenase Gene
Molecular pharmacology, 1998Co-Authors: Yong Tai Hou, Hsueh Kung Lin, Trevor M. PenningAbstract:Rat liver 3alpha-hydroxysteroid/Dihydrodiol Dehydrogenase (3alpha-HSD/DD), a member of the aldo-keto reductase superfamily, inactivates circulating steroid hormones and may contribute to the carcinogenicity of polycyclic aromatic hydrocarbons (PAHs) by oxidizing trans-Dihydrodiols to reactive o-quinones with the concomitant generation of reactive oxygen species. The 3alpha-HSD/DD gene has been cloned, and its 5'-flanking region contains a negative response element (NRE; -797 to -498 bp) that may repress constitutive expression by binding to Oct transcription factors. Upstream from the NRE are three distal imperfect glucocorticoid response elements (GRE1, GRE2, and GRE3); in addition, a proximal imperfect GRE (GRE4) is adjacent to an Oct binding site in the NRE. When rat hepatocytes were cultured on Matrigel and exposed to dexamethasone (Dex), steady state levels of 3alpha-HSD/DD mRNA were increased 4-fold in a dose-dependent manner, yielding an EC50 value of 10 nM. Time to maximal response was 24 hr, and the effect was blocked with the anti-glucocorticoid RU486. Measurement of the half-life of 3alpha-HSD/DD mRNA, with and without Dex treatment, indicated that the increase in steady state mRNA levels was not due to increased mRNA stability. By contrast, nuclear run-off experiments using nuclei obtained from Dex-stimulated hepatocytes indicated that Dex increased transcription of the rat 3alpha-HSD/DD gene. Tandem repeats of the imperfect GRE1, GRE2, GRE3, and GRE4 were inserted into thymidine kinase-chloramphenicol acetyl-transferase vectors and cotransfected with the human glucocorticoid receptor into human hepatoma cells. On treatment with Dex, maximal trans-activation of the chloramphenicol acetyl-transferase reporter gene activity was mediated via the proximal GRE (GRE4). These data imply that GRE4 is a functional cis-element and that binding of the occupied glucocorticoid receptor to this element increases 3alpha-HSD/DD gene transcription. A model is proposed for the positive and negative regulation of the rat 3alpha-HSD/DD gene by the glucocorticoid receptor and Oct transcription factors, respectively.
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Disposition and Biological Activity of Benzo[a]pyrene-7,8-dione. A Genotoxic Metabolite Generated by Dihydrodiol Dehydrogenase†,‡
Biochemistry, 1996Co-Authors: Lynn Flowers, Michael E. Burczynski, Ronald G. Harvey, Wendy F. Bleczinski, Trevor M. PenningAbstract:A novel pathway of polycyclic aromatic hydrocarbon metabolism involves the oxidation of non-K-region trans-Dihydrodiols to yield o-quinones, a reaction catalyzed by Dihydrodiol Dehydrogenase (DD). We have recently shown that in isolated rat hepatocytes (±)-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene (BP-diol) was oxidized by this route to yield benzo[a]pyrene-7,8-dione (BPQ). We now report the disposition of BPQ and its mutagenic and genotoxic properties. Using [3H]BPQ it was found that 30% of the radioactivity was sequestered by rat hepatocytes into the cell pellet. Isolation of hepatocyte DNA provided evidence for a low level of covalent incorporation of BPQ into DNA (30 ± 17 adducts/106 base pairs). Examination of the hepatocellular DNA by agarose gel electrophoresis following treatment with BPQ indicated that extensive fragmentation had occurred. DNA fragmentation was also observed when hepatocytes were treated with BP-diol and this effect was attenuated by indomethacin, a DD inhibitor. Hepatocytes ...
Yukuhiko Asada - One of the best experts on this subject based on the ideXlab platform.
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Structure of monkey dimeric Dihydrodiol Dehydrogenase in complex with isoascorbic acid.
Acta crystallographica. Section D Biological crystallography, 2008Co-Authors: Vincenzo Carbone, Akira Hara, Rie Sumii, Shuhei Ishikura, Yukuhiko Asada, Ossama El-kabbaniAbstract:Mammalian dimeric Dihydrodiol Dehydrogenase (DD) is identical to NADP+-dependent D-xylose Dehydrogenase. A recent investigation showed that the three-dimensional structure of monkey DD is similar to those of prokaryotic NADP(H)-dependent glucose-fructose oxidoreductase (GFO) and 1,5-anhydro-D-fructose reductase (AFR); however, it differs in coenzyme-binding and catalytic residues. Dimeric DD has a high affinity for NADP(H) when compared with AFR and differs from both GFO and AFR in its specificity for sugars and hydrophobic xenobiotic compounds as substrates. The crystal structure of monkey dimeric DD complexed with the inhibitor isoascorbic acid has been determined at 2.59 angstroms resolution. Molecular modelling of coenzyme binding complemented with site-directed mutagenesis has been utilized to propose a binding mode for the coenzyme molecule and to gain insights into the roles of the residues comprising the active site and coenzyme-binding domain of DD. Several key residues have been identified within the coenzyme-binding domain, including Arg37, Arg41, His76 and His79, that contribute to the high affinity for coenzyme. The interaction of Arg37 and Arg41 with the 2'-phosphate and adenine-ring moiety of the coenzyme has been established from the large increases (29-fold to 438-fold) in the Kd values for NADP(H) for the R37D and R41D mutant enzymes. The mutation of several residues lining the inhibitor-binding site of DD suggested the involvement of Trp125, Phe154, Trp254 and Phe279 in determining the broad substrate specificity and inhibitor potency of the enzyme. In addition, mutants of Lys97, which is present near the catalytic residue Tyr180, greatly reduced the kcat value without changing the Kd values for coenzyme, suggesting the importance of Lys97 in the catalytic mechanism of DD.
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Crystallization and preliminary X-ray diffraction analysis of monkey dimeric Dihydrodiol Dehydrogenase
Acta Crystallographica Section D Biological Crystallography, 2001Co-Authors: Ossama El-kabbani, Yukuhiko Asada, Roland P.-t. Chung, Paul A. Ramsland, Syuhei Ishikura, Noriyuki Usami, Akira HaraAbstract:Dihydrodiol Dehydrogenase catalyzes the NADP+-linked oxidation of trans-Dihydrodiols of aromatic hydrocarbons to corresponding catechols and exists in multiple forms in mammalian tissues. The dimeric form of mammalian Dihydrodiol Dehydrogenase has a primary structure distinct from the previously known mammalian enzymes and may constitute a novel protein family with the prokaryotic proteins. Monkey kidney dimeric Dihydrodiol Dehydrogenase was crystallized from buffered ammonium phosphate solution using the hanging-drop vapour-diffusion method. The crystals diffract to 2.65 A resolution in the laboratory and belong to the hexagonal P6122 or P6522 space group, with unit-cell parameters a = b = 122.8, c = 121.3 A, α = β = 90, γ = 120°.
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Crystallization and preliminary X-ray diffraction analysis of monkey dimeric Dihydrodiol Dehydrogenase.
Acta crystallographica. Section D Biological crystallography, 2001Co-Authors: Ossama El-kabbani, Yukuhiko Asada, Roland P.-t. Chung, Paul A. Ramsland, Syuhei Ishikura, Noriyuki Usami, Akira HaraAbstract:Dihydrodiol Dehydrogenase catalyzes the NADP(+)-linked oxidation of trans-Dihydrodiols of aromatic hydrocarbons to corresponding catechols and exists in multiple forms in mammalian tissues. The dimeric form of mammalian Dihydrodiol Dehydrogenase has a primary structure distinct from the previously known mammalian enzymes and may constitute a novel protein family with the prokaryotic proteins. Monkey kidney dimeric Dihydrodiol Dehydrogenase was crystallized from buffered ammonium phosphate solution using the hanging-drop vapour-diffusion method. The crystals diffract to 2.65 A resolution in the laboratory and belong to the hexagonal P6(1)22 or P6(5)22 space group, with unit-cell parameters a = b = 122.8, c = 121.3 A, alpha = beta = 90, gamma = 120 degrees.
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Roles of His-79 and Tyr-180 of D-xylose/Dihydrodiol Dehydrogenase in catalytic function.
Biochemical and biophysical research communications, 2000Co-Authors: Yukuhiko Asada, Syuhei Ishikura, Noriyuki Usami, Shinya Aoki, Akira HaraAbstract:Abstract Mammalian dimeric Dihydrodiol Dehydrogenase is identical with d -xylose Dehydrogenase and belongs to a protein family with prokaryotic proteins including glucose-fructose oxidoreductase. Of the conserved residues in this family, either His-79 or Tyr-180 of d -xylose/Dihydrodiol Dehydrogenase has been proposed to be involved in the catalytic function. Site-directed mutagenesis was used to examine the roles of the two residues of the monkey enzyme. A mutant, Y180F, was almost inactive, but, similarly to the wild-type enzyme, exhibited high affinity for NADP(H) and fluorescence energy transfer upon binding of NADPH. The H79Q mutation had kinetically largest effects on Kd (>7-fold increase) and Km (>25-fold increase) for NADP(H), and eliminated the fluorescence energy transfer. Interestingly, the Dehydrogenase activity of this mutant was potently inhibited with a 190-fold increase in the Km for NADP+ by high ionic strength, which activated the activity of the wild-type enzyme. These results suggest a critical role of Tyr-180 in the catalytic function of this class of enzymes, in addition to functions of His-79 in the coenzyme binding and chemical steps of the reaction.
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roles of his 79 and tyr 180 of d xylose Dihydrodiol Dehydrogenase in catalytic function
Biochemical and Biophysical Research Communications, 2000Co-Authors: Yukuhiko Asada, Syuhei Ishikura, Noriyuki Usami, Shinya Aoki, Akira HaraAbstract:Abstract Mammalian dimeric Dihydrodiol Dehydrogenase is identical with d -xylose Dehydrogenase and belongs to a protein family with prokaryotic proteins including glucose-fructose oxidoreductase. Of the conserved residues in this family, either His-79 or Tyr-180 of d -xylose/Dihydrodiol Dehydrogenase has been proposed to be involved in the catalytic function. Site-directed mutagenesis was used to examine the roles of the two residues of the monkey enzyme. A mutant, Y180F, was almost inactive, but, similarly to the wild-type enzyme, exhibited high affinity for NADP(H) and fluorescence energy transfer upon binding of NADPH. The H79Q mutation had kinetically largest effects on Kd (>7-fold increase) and Km (>25-fold increase) for NADP(H), and eliminated the fluorescence energy transfer. Interestingly, the Dehydrogenase activity of this mutant was potently inhibited with a 190-fold increase in the Km for NADP+ by high ionic strength, which activated the activity of the wild-type enzyme. These results suggest a critical role of Tyr-180 in the catalytic function of this class of enzymes, in addition to functions of His-79 in the coenzyme binding and chemical steps of the reaction.
T M Penning - One of the best experts on this subject based on the ideXlab platform.
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genomic structure of rat 3α hydroxysteroid Dihydrodiol Dehydrogenase 3α hsd dd akr1c9
The Journal of Steroid Biochemistry and Molecular Biology, 1999Co-Authors: Hsueh Kung Lin, Chien Fu Hung, Margaret Moore, T M PenningAbstract:Rat liver 3alpha-hydroxysteroid/Dihydrodiol Dehydrogenase (3alpha-HSD/DD) is a member of the aldo-keto reductase (AKR) superfamily. It is involved in the inactivation of steroid hormones and the metabolic activation of polycyclic aromatic hydrocarbons (PAH) by converting trans-Dihydrodiols into reactive and redox-active o-quinones. The structure of the 5'-flanking region of the gene and factors involved in the constitutive and regulated expression of this gene have been reported [H.-K. Lin, T.M. Penning, Cloning, sequencing, and functional analysis of the 5'-flanking region of the rat 3alpha-hydroxysteroid/Dihydrodiol Dehydrogenase gene, Cancer Res. 55 (1995) 4105-4113]. We now describe the complete genomic structure of the rat type 1 3alpha-HSD/DD gene. Charon 4A and P1 genomic clones contained at least three rat genes (type 1, type 2 and type 3 3alpha-HSD/DD) each of which encoded for the same open reading frame (ORF) but differed in their exon-intron organization. 5'-RACE confirmed that the type 1 3alpha-HSD/DD gene encodes for the dominant transcript in rat liver and it was the regulation of this gene that was previously studied. The rat type 1 3alpha-HSD/DD gene is 30 kb in length and consists of nine exons and eight introns. Exon 9 encodes +931 to 966 bp of the ORF and the 1292 bp 3'-UTR implicated in mRNA stability. This genomic structure is nearly identical to the homologous human genes, type 1 3alpha-HSD (chlordecone reductase/DD4, AKR1C4), type 2 3alpha-HSD (AKR1C3) and type 3 3alpha-HSD (bile-acid binding protein, AKR1C2) genes. Three different cDNA's containing identical ORFs for 3alpha-HSD have been reported suggesting that all three genes may be expressed in rat liver. Using 5' primers corresponding to the 5'-UTR's of the three different cDNA's only one PCR fragment was obtained and corresponded to the type 1 3alpha-HSD/DD gene. These data suggested that the type 2 and type 3 3alpha-HSD/DD genes are not abundantly expressed in rat liver. It is unknown whether the type 2 and type 3 3alpha-HSD/DD genes represent pseudo-genes or whether they represent genes that are differentially expressed in other rat tissues.
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Genomic structure of rat 3alpha-hydroxysteroid/Dihydrodiol Dehydrogenase (3alpha-HSD/DD, AKR1C9).
The Journal of steroid biochemistry and molecular biology, 1999Co-Authors: Hsueh Kung Lin, Chien Fu Hung, Margaret Moore, T M PenningAbstract:Rat liver 3alpha-hydroxysteroid/Dihydrodiol Dehydrogenase (3alpha-HSD/DD) is a member of the aldo-keto reductase (AKR) superfamily. It is involved in the inactivation of steroid hormones and the metabolic activation of polycyclic aromatic hydrocarbons (PAH) by converting trans-Dihydrodiols into reactive and redox-active o-quinones. The structure of the 5'-flanking region of the gene and factors involved in the constitutive and regulated expression of this gene have been reported [H.-K. Lin, T.M. Penning, Cloning, sequencing, and functional analysis of the 5'-flanking region of the rat 3alpha-hydroxysteroid/Dihydrodiol Dehydrogenase gene, Cancer Res. 55 (1995) 4105-4113]. We now describe the complete genomic structure of the rat type 1 3alpha-HSD/DD gene. Charon 4A and P1 genomic clones contained at least three rat genes (type 1, type 2 and type 3 3alpha-HSD/DD) each of which encoded for the same open reading frame (ORF) but differed in their exon-intron organization. 5'-RACE confirmed that the type 1 3alpha-HSD/DD gene encodes for the dominant transcript in rat liver and it was the regulation of this gene that was previously studied. The rat type 1 3alpha-HSD/DD gene is 30 kb in length and consists of nine exons and eight introns. Exon 9 encodes +931 to 966 bp of the ORF and the 1292 bp 3'-UTR implicated in mRNA stability. This genomic structure is nearly identical to the homologous human genes, type 1 3alpha-HSD (chlordecone reductase/DD4, AKR1C4), type 2 3alpha-HSD (AKR1C3) and type 3 3alpha-HSD (bile-acid binding protein, AKR1C2) genes. Three different cDNA's containing identical ORFs for 3alpha-HSD have been reported suggesting that all three genes may be expressed in rat liver. Using 5' primers corresponding to the 5'-UTR's of the three different cDNA's only one PCR fragment was obtained and corresponded to the type 1 3alpha-HSD/DD gene. These data suggested that the type 2 and type 3 3alpha-HSD/DD genes are not abundantly expressed in rat liver. It is unknown whether the type 2 and type 3 3alpha-HSD/DD genes represent pseudo-genes or whether they represent genes that are differentially expressed in other rat tissues.
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dexamethasone regulation of the rat 3α hydroxysteroid Dihydrodiol Dehydrogenase gene
Molecular Pharmacology, 1998Co-Authors: Yong Tai Hou, Hsueh Kung Lin, T M PenningAbstract:Rat liver 3alpha-hydroxysteroid/Dihydrodiol Dehydrogenase (3alpha-HSD/DD), a member of the aldo-keto reductase superfamily, inactivates circulating steroid hormones and may contribute to the carcinogenicity of polycyclic aromatic hydrocarbons (PAHs) by oxidizing trans-Dihydrodiols to reactive o-quinones with the concomitant generation of reactive oxygen species. The 3alpha-HSD/DD gene has been cloned, and its 5'-flanking region contains a negative response element (NRE; -797 to -498 bp) that may repress constitutive expression by binding to Oct transcription factors. Upstream from the NRE are three distal imperfect glucocorticoid response elements (GRE1, GRE2, and GRE3); in addition, a proximal imperfect GRE (GRE4) is adjacent to an Oct binding site in the NRE. When rat hepatocytes were cultured on Matrigel and exposed to dexamethasone (Dex), steady state levels of 3alpha-HSD/DD mRNA were increased 4-fold in a dose-dependent manner, yielding an EC50 value of 10 nM. Time to maximal response was 24 hr, and the effect was blocked with the anti-glucocorticoid RU486. Measurement of the half-life of 3alpha-HSD/DD mRNA, with and without Dex treatment, indicated that the increase in steady state mRNA levels was not due to increased mRNA stability. By contrast, nuclear run-off experiments using nuclei obtained from Dex-stimulated hepatocytes indicated that Dex increased transcription of the rat 3alpha-HSD/DD gene. Tandem repeats of the imperfect GRE1, GRE2, GRE3, and GRE4 were inserted into thymidine kinase-chloramphenicol acetyl-transferase vectors and cotransfected with the human glucocorticoid receptor into human hepatoma cells. On treatment with Dex, maximal trans-activation of the chloramphenicol acetyl-transferase reporter gene activity was mediated via the proximal GRE (GRE4). These data imply that GRE4 is a functional cis-element and that binding of the occupied glucocorticoid receptor to this element increases 3alpha-HSD/DD gene transcription. A model is proposed for the positive and negative regulation of the rat 3alpha-HSD/DD gene by the glucocorticoid receptor and Oct transcription factors, respectively.
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disposition and biological activity of benzo a pyrene 7 8 dione a genotoxic metabolite generated by Dihydrodiol Dehydrogenase
Biochemistry, 1996Co-Authors: Lynn Flowers, Michael E. Burczynski, Ronald G. Harvey, Wendy F. Bleczinski, T M PenningAbstract:A novel pathway of polycyclic aromatic hydrocarbon metabolism involves the oxidation of non-K-region trans-Dihydrodiols to yield o-quinones, a reaction catalyzed by Dihydrodiol Dehydrogenase (DD). We have recently shown that in isolated rat hepatocytes (±)-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene (BP-diol) was oxidized by this route to yield benzo[a]pyrene-7,8-dione (BPQ). We now report the disposition of BPQ and its mutagenic and genotoxic properties. Using [3H]BPQ it was found that 30% of the radioactivity was sequestered by rat hepatocytes into the cell pellet. Isolation of hepatocyte DNA provided evidence for a low level of covalent incorporation of BPQ into DNA (30 ± 17 adducts/106 base pairs). Examination of the hepatocellular DNA by agarose gel electrophoresis following treatment with BPQ indicated that extensive fragmentation had occurred. DNA fragmentation was also observed when hepatocytes were treated with BP-diol and this effect was attenuated by indomethacin, a DD inhibitor. Hepatocytes ...
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structure of 3 alpha hydroxysteroid Dihydrodiol Dehydrogenase complexed with nadp
Biochemistry, 1996Co-Authors: Melanie J. Bennett, T M Penning, Brian P. Schlegel, Joseph M. Jez, Mitchell LewisAbstract:Rat liver 3α-hydroxysteroid/Dihydrodiol Dehydrogenase (3α-HSD) inactivates circulating steroid hormones and is involved in polycyclic aromatic hydrocarbon (PAH) carcinogenesis. It is the only HSD of known structure in the aldo−keto reductase (AKR) superfamily and may provide a paradigm for other mammalian HSDs in this family. The structure of the 3α-HSD·NADP+ binary complex has been determined at 2.7 A resolution and refined to a crystallographic R-factor of 23.4% with good geometry. The model is similar to other binary complexes in the AKR superfamily in that NADP+ binds at the C-terminal end of an α/β barrel. However, it is unique in that NADP+ is bound in two alternate conformations, probably because of the lack of a salt-linked “safety belt” over the pyrophosphate bridge. The structure supports a previously proposed catalytic mechanism for carbonyl reduction in which Tyr 55 is the general acid, and its effective pKa is lowered by the adjacent Lys 84. We present evidence that the structurally distinct ...