Aldehyde reductase is a major protein associated with 3-deoxyglucosone reductase activity in rat, pig and human livers.
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ABSTRACT:
SUBMITTER: Kanazu T
PROVIDER: S-EPMC1151532 | biostudies-other | 1991 Nov
REPOSITORIES: biostudies-other
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Project description:The reduction of p-nitroso-N-dimethylaniline, p-nitroso-N-diethylaniline, p-nitrosophenol and p-nitroso-N-phenylaniline with NADPH in the presence of aldehyde reductases 1 and 2 is described. The reactivity of these nitroso substrates is increased by hydrophobic substituents and those promoting OH- elimination from the molecule of the reduced substrate. NN-Dimethylbenzoquinonedi-iminium cation was proved to be the reaction product formed from p-nitroso-N-dimethylaniline. The kinetics of the reduction of p-nitroso-N-dimethylaniline catalysed with aldehyde reductase 1 are rather complex at pH 7, and the preferred-pathway mechanism is probably involved. The reaction sequence approaches the ordered pattern at pH 8.5. It was shown that NADPH in equilibrium NADP+ recyclization proceeds in the presence of NADP+, p-nitroso-N-dimethylaniline, cyclohexanol and aldehyde reductase 1, the alcohol oxidation being the slowest step in this reaction. However, the rate of cyclohexanol oxidation surpasses that of the dissociation of NADPH from the enzyme.
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Project description:Advances in synthetic biology and metabolic engineering have enabled the construction of novel biological routes to valuable chemicals using suitable microbial hosts. Aldehydes serve as chemical feedstocks in the synthesis of rubbers, plastics, and other larger molecules. Microbial production of alkanes is dependent on the formation of a fatty aldehyde intermediate which is converted to an alkane by an aldehyde deformylating oxygenase (ADO). However, microbial hosts such as Escherichia coli are plagued by many highly active endogenous aldehyde reductases (ALRs) that convert aldehydes to alcohols, which greatly complicates strain engineering for aldehyde and alkane production. It has been shown that the endogenous ALR activity outcompetes the ADO enzyme for fatty aldehyde substrate. The large degree of ALR redundancy coupled with an incomplete database of ALRs represents a significant obstacle in engineering E. coli for either aldehyde or alkane production. In this study, we identified 44 ALR candidates encoded in the E. coli genome using bioinformatics tools, and undertook a comprehensive screening by measuring the ability of these enzymes to produce isobutanol. From the pool of 44 candidates, we found five new ALRs using this screening method (YahK, DkgA, GldA, YbbO, and YghA). Combined deletions of all 13 known ALRs resulted in a 90-99% reduction in endogenous ALR activity for a wide range of aldehyde substrates (C2-C12). Elucidation of the ALRs found in E. coli could guide one in reducing competing alcohol formation during alkane or aldehyde production.
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Project description:Aldehyde reductase was purified from pig kidney cortex to homogeneity by a new procedure. The molecular weight of the enzyme was estimated by sedimentation equilibrium to be 43 700 and by gel electrophoresis in the presence of sodium dodecyl sulphate to be 41 700. The enzyme is clearly a monomer. The enzyme preparation contained no significant quantities of zinc, manganese or copper and had no essential histidine or thiol groups. Changes in the absorption and fluorescence spectra of NADPH were observed on formation of the enzyme-NADPH complexes. Large changes in the fluorescence spectra were also observed in the presence of sodium barbitone or Warfarin. These changes were used as the basis of active-site titrations, which showed that the enzyme had one active site per molecule. The dissociation constants of NADPH and NADP+ from binary complexes with the enzyme were estimated in spectrophotometric titrations.
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Project description:The masking of charged amino or carboxy groups by N-phthalidylation and O-phthalidylation has been used to improve the absorption of many drugs, including ampicillin and 5-fluorouracil. Following absorption of such prodrugs, the phthalidyl group is hydrolysed to release 2-carboxybenzaldehyde (2-CBA) and the pharmaceutically active compound; in humans, 2-CBA is further metabolized to 2-hydroxymethylbenzoic acid by reduction of the aldehyde group. In the present work, the enzyme responsible for the reduction of 2-CBA in humans is identified as a homologue of rat aflatoxin B1-aldehyde reductase (rAFAR). This novel human aldo-keto reductase (AKR) has been cloned from a liver cDNA library, and together with the rat protein, establishes the AKR7 family of the AKR superfamily. Unlike its rat homologue, human AFAR (hAFAR) appears to be constitutively expressed in human liver, and is widely expressed in extrahepatic tissues. The deduced human and rat protein sequences share 78% identity and 87% similarity. Although the two AKR7 proteins are predicted to possess distinct secondary structural features which distinguish them from the prototypic AKR1 family of AKRs, the catalytic- and NADPH-binding residues appear to be conserved in both families. Certain of the predicted structural features of the AKR7 family members are shared with the AKR6 beta-subunits of voltage-gated K+-channels. In addition to reducing the dialdehydic form of aflatoxin B1-8,9-dihydrodiol, hAFAR shows high affinity for the gamma-aminobutyric acid metabolite succinic semialdehyde (SSA) which is structurally related to 2-CBA, suggesting that hAFAR could function as both a SSA reductase and a 2-CBA reductase in vivo. This hypothesis is supported in part by the finding that the major peak of 2-CBA reductase activity in human liver co-purifies with hAFAR protein.
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Project description:BackgroundLiver perfusates exhibit theoretical advantages regarding the discovery of disease biomarkers because they contain proteins that readily enter the blood-stream, and perfusion preserves the disease state in its natural context. The purpose of the study is to explore the value of liver perfusate proteome in the biomarker discovery of liver diseases.ResultsIn this study, 86 differentially expressed proteins were identified in perfusates from isolated rat livers metastasized by Walker-256 tumor cells. Among these proteins, 27 were predicted to be secreted, and 59 were intracellular or membrane proteins. Most of the secretory proteins (70.4%) were decreased in metastasized liver perfusates. The main canonical ingenuity pathway to which these secretory proteins belonged was acute phase response, which indicated that the liver-associated immune reaction was damaged by the metastasis. In contrast, most of the intracellular or membrane proteins (86.4%) exhibited higher relative abundances in the metastasized liver perfusates. Some of these proteins, including Rpl21, Atic, Eif3s2, Echs1, Eps15 and Ywhab, have previously been reported to be involved in cancer genesis and progression. As a member of the 14-3-3 protein family, Ywhab plays a key role in cellular proliferation and oncogenic transformation and has been reported to be involved in the development of breast cancer. Its abundance was elevated by 3.5-fold in the metastasized perfusates. Validation by Western blotting revealed a 3.7-fold increase in the abundance of this protein in metastasized plasma.ConclusionsThese results show that perfusate proteome can be used as an alternative initial resource for biomarker identification, which ultimately requires validation in serum.
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Project description:We have kinetically and immunologically demonstrated that testosterone 17 beta-dehydrogenase (NADP+) isoenzymes (EC 1.1.1.64) and aldehyde reductase (EC 1.1.1.2) from guinea-pig liver catalyse the oxidation of benzene dihydrodiol (trans-1,2-dihydroxycyclohexa-3,5-diene) to catechol. One isoenzyme of testosterone 17 beta-dehydrogenase, which has specificity for 5 beta-androstanes, oxidized benzene dihydrodiol at a 3-fold higher rate than 5 beta-dihydrotestosterone, and showed a more than 4-fold higher affinity for benzene dihydrodiol and Vmax. value than did another isoenzyme, which exhibits specificity for 5 alpha-androstanes, and aldehyde reductase. Immunoprecipitation of guinea-pig liver cytosol with antisera against the testosterone 17 beta-dehydrogenase isoenzymes and aldehyde reductase indicated that most of the benzene dihydrodiol dehydrogenase activity in the tissue is due to testosterone 17 beta-dehydrogenase.
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Project description:Initial-rate measurements were made of the oxidations of pyridine-3-methanol and glycerol by NADP+ and of the reduction of the corresponding aldehydes by NADPH catalysed by pig kidney aldehyde reductase. In addition, a brief survey of the specificity of the enzyme towards aldehyde substrates and its sensitivity to the inhibitors ethacrynic acid, sodium barbitone and warfarin was made. The detailed kinetic work indicates a compulsory mechanism for aldehyde reduction, with NADPH binding before aldehyde. For alcohol oxidation, however, it is necessary to postulate the formation of kinetically significant amounts of binary complexes of the type enzyme-alcohol to explain the results. Thus, for alcohol oxidation random-order addition of substrates may occur. Inhibition studies of the kinetics of aldehyde reduction in the presence of the corresponding alcohol product provide further evidence for the existence of enzyme-alcohol complexes. Finally, detailed kinetic studies were made of the inhibition of pyridine-3-aldehyde reduction by sodium barbitone. The mechanism of the inhibition is discussed.
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