Dimeric dihydrodiol dehydrogenase is an efficient primate 1,5-anhydro-D-fructose reductase.

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.

Sibutramine provokes apoptosis of aortic endothelial cells through altered production of reactive oxygen and nitrogen species.

Overdose administration of sibutramine, a serotonin-noradrenalin reuptake inhibitor, is considered to elicit severe side effects including hypertension, whose pathogenic mechanism remains unclear. Here, we found that 48-h incubation with >10µM sibutramine provokes apoptosis of human aortic endothelial (HAE) cells. Treatment with the lethal concentration of sibutramine facilitated production of reactive oxygen species (ROS), altered expression of endoplasmic reticulum stress response genes (heat shock protein 70 and C/EBP homologous protein), and inactivated 26S proteasome-based proteolysis. The treatment also decreased cellular level of nitric oxide (NO) through lowering of expression and activity of endothelial NO synthase. These results suggest that ROS production and depletion of NO are crucial events in the apoptotic mechanism and may be linked to the pathogenesis of vasoconstriction elicited by the drug. Compared to sibutramine, its metabolites (N-desmethylsibutramine and N-didesmethylsibutramine) were much less cytotoxic to HAE cells, which hardly metabolized sibutramine. In contrast, both the drug and metabolites showed low cytotoxicity to hepatic HepG2 cells with high metabolic potency and expression of cytochrome P450 (CYP) 3A4. The cytotoxicity of sibutramine to HepG2 and Chang Liver cells was remarkably augmented by inhibition and knockdown of CYP3A4. This study also suggests an inverse relationship between sibutramine cytotoxicity and CYP3A4-mediated metabolism into the N-desmethyl metabolites.

Enhancement of Endothelial Barrier Permeability by Mitragynine.

Persistent inhalation of mitragynine (MG), a major alkaloid in the leaves of Mitragyna speciosa, causes various systemic adverse effects such as seizure, diarrhea and arthralgias, but its toxicity to endothelial cells and effects on barrier function of the cells are poorly understood. In this study, we compared toxicities of MG and mitraphylline, another constituent of the leaves, against human aortic endothelial (HAE), bronchial BEAS-2B, neuronal SK-N-SH, hepatic HepG2, kidney HEK293, gastric MKN45, colon DLD1, lung A549, breast MCF7 and prostate LNCaP cells, and found that MG, but not mitraphylline, shows higher toxicity to HAE cells compared to the other cells. Forty-eight-hours incubation of HAE cells with a high concentration of MG (60 µM) provoked apoptotic cell death, which was probably due to signaling through enhanced reactive oxygen species (ROS) generation and resultant caspase activation. Treatment of the cells with MG at sublethal concentrations less than 20 µM significantly lowered transendothelial electrical resistance and elevated paracellular permeability, without affecting the cell viability. In addition, the MG-elicited lowering of the resistance was abolished by a ROS inhibitor N-acetyl-L-cysteine and augmented by H2O2 and 9,10-phenanthrenequinone, which generates ROS through its redox cycle. These results suggest the contribution of ROS generation to the increase in endothelial barrier permeability.

Inhibition of aldo-keto reductase family 1 member B10 by unsaturated fatty acids.

A human member of the aldo-keto reductase (AKR) superfamily, AKR1B10, is a cytosolic NADPH-dependent reductase toward various carbonyl compounds including reactive aldehydes, and is normally expressed in intestines. The enzyme is overexpressed in several extraintestinal cancers, and suggested as a potential target for cancer treatment. We found that saturated and cis-unsaturated fatty acids inhibit AKR1B10. Among the saturated fatty acids, myristic acid was the most potent, showing the IC50 value of 4.2 µM cis-Unsaturated fatty acids inhibited AKR1B10 more potently, and linoleic, arachidonic, and docosahexaenoic acids showed the lowest IC50 values of 1.1 µM. The inhibition by these fatty acids was reversible and kinetically competitive with respect to the substrate, showing the Ki values of 0.24-1.1 µM. These fatty acids, except for α-linoleic acid, were much less inhibitory to structurally similar aldose reductase. Site-directed mutagenesis study suggested that the fatty acids interact with several active site residues of AKR1B10, of which Gln114, Val301 and Gln303 are responsible for the inhibitory selectivity. Linoleic and arachidonic acids also effectively inhibited AKR1B10-mediated 4-oxo-2-nonenal metabolism in HCT-15 cells. Thus, the cis-unsaturated fatty acids may be used as an adjuvant therapy for treatment of cancers that up-regulate AKR1B10.

Identification of a determinant for strict NADP(H)-specificity and high sensitivity to mixed-type steroid inhibitor of rabbit aldo-keto reductase 1C33 by site-directed mutagenesis.

In rabbit tissues, hydroxysteroid dehydrogenase belonging to the aldo-keto reductase (AKR) superfamily exists in six isoforms (AKRs: 1C5 and 1C29-1C33), sharing >73% amino acid sequence identity. AKR1C33 is strictly NADPH-specific, in contrast to dual NADPH/NADH specificity of the other isoforms. All coenzyme-binding residues of the structurally elucidated AKR1C5 are conserved in other isoforms, except that S217 (interacting with the pyrophosphate moiety) and T273 (interacting with the 2'-phosphate moiety) are replaced with F217 and N272, respectively, in AKR1C33. To explore the determinants for the NADPH specificity of AKR1C33, we prepared its F217S and N272T mutant enzymes. The mutation of F217S, but not N272T, converted AKR1C33 into a dually coenzyme-specific form that showed similar kcat values for NAD(P)H to those of AKR1C32. The reverse mutation (S217F) in dually coenzyme-specific AKR1C32 produced a strictly NADPH-specific form. The F217S mutation also abolished the activity towards 3-keto-5ß-cholestanes that are substrates specific to AKR1C33, and markedly decreased the sensitivity to 4-pregnenes (such as deoxycorticosterone and medroxyprogesterone acetate) that were found to be potent mixed-type inhibitors of the wild-type enzyme. The results indicate the important role of F217 in the strict NADPH-dependency, as well as its involvement in the unique catalytic properties of AKR1C33.

Synthesis of 8-hydroxy-2-iminochromene derivatives as selective and potent inhibitors of human carbonyl reductase 1.

Human carbonyl reductase 1 (CBR1), a member of the short-chain dehydrogenase/reductase superfamily, reduces anthracycline anticancer drugs to their less potent anticancer C-13 hydroxy metabolites, which are linked with pathogenesis of cardiotoxicity, a side effect of the drugs. CBR1 inhibitors are thought to be promising agents for adjuvant therapy with a twofold beneficial effect in prolonging the anticancer efficacy of the anthracyclines while decreasing cardiotoxicity. In order to search for new potential inhibitors of CBR1, we synthesized a series of des-methoxyphenyl derivatives of (Z)-2-(4-methoxyphenylimino)-7-hydroxy-N-(pyridin-2-yl)-2H-chromene-3-carboxamide (1) that was developed previously as a potent inhibitor of aldo-keto reductase (AKR) 1B10 and AKR1B1. Among the newly synthesized inhibitors, 8-hydroxy-2-imino-2H-chromene-3-carboxylic acid (2-chlorophenyl)amide (13h) was the most potent competitive inhibitor of CBR1, showing a Ki value of 15 nM. 13h also showed high selectivity to CBR1 over its isozyme CBR3 and other enzymes with CBR activity (AKR1B1, AKR1B10, AKR1C1, AKR1C2, AKR1C4, DXCR and DHRS4). Furthermore, 13h inhibited the cellular metabolism by CBR1 at its concentration of 4 µM. The structure-activity relationship of the derivatives, site-directed mutagenesis of putative binding residues (Met141 and Trp229) and molecular docking of 13h in CBR1 revealed that the interactions of 13h with the substrate-binding residues (Ser139, Met141, Tyr193 and Trp229) are important for the tight binding.

Up-Regulation of Carbonyl Reductase 1 Renders Development of Doxorubicin Resistance in Human Gastrointestinal Cancers.

Doxorubicin (DOX) is widely used for the treatment of a wide range of cancers such as breast and lung cancers, and malignant lymphomas, but is generally less efficacious in gastrointestinal cancers. The most accepted explanation for the DOX refractoriness is its resistance development. Here, we established DOX-resistant phenotypes of human gastric MKN45 and colon LoVo cells by continuous exposure to incremental concentrations of the drug. While the parental MKN45 and LoVo cells expressed carbonyl reductase 1 (CBR1) highly and moderately, respectively, the gain of DOX resistance further elevated the CBR1 expression. Additionally, the DOX-elicited cytotoxicity was lowered by overexpression of CBR1 and inversely strengthened by knockdown of the enzyme using small interfering RNA or pretreating with the specific inhibitor quercetin, which also reduced the DOX refractoriness of the two resistant cells. These suggest that CBR1 is a key enzyme responsible for the DOX resistance of gastrointestinal cancer cells and that its inhibitor is useful in the adjuvant therapy. Although CBR1 is known to metabolize DOX to a less toxic anticancer metabolite doxorubicinol, its overexpression in the parental cells hardly show significant reductase activity toward low concentration of DOX. In contrast, the overexpression of CBR1 increased the reductase activity toward an oxidative stress-derived cytotoxic aldehyde 4-oxo-2-nonenal. The sensitivity of the DOX-resistant cells to 4-oxo-2-nonenal was lower than that of the parental cells, and the resistance-elicited hyposensitivity was almost completely ameliorated by addition of the CBR1 inhibitor. Thus, CBR1 may promote development of DOX resistance through detoxification of cytotoxic aldehydes, rather than the drug's metabolism.

Cloning and characterization of four rabbit aldo-keto reductases featuring broad substrate specificity for xenobiotic and endogenous carbonyl compounds: relationship with multiple forms of drug ketone reductases.

Multiple forms of reductases for several drug ketones were isolated from rabbit liver, but their interrelationship and physiologic roles remain unknown. We isolated cDNAs for four aldo-keto reductases (AKR1C30, AKR1C31, AKR1C32, and AKR1C33), which share high amino acid sequence identity with the partial sequences of two rabbit naloxone reductases. The four recombinant enzymes reduced a variety of carbonyl compounds, including endogenous α-dicarbonyls (e.g., isatin and diacetyl), aldehydes (e.g., farnesal and 4-oxo-2-nonenal), and ketosteroids. They differed in specificity for drug ketones and ketosteroids. Although daunorubicin and befunolol were common substrates of all of the enzymes, AKR enzymes specifically reduced naloxone (AKR1C30, AKR1C32, and AKR1C33), metyrapone (AKR1C32 and AKR1C33), loxoprofen (AKR1C31 and AKR1C32), ketotifen (AKR1C30), and naltrexone and fenofibric acid (AKR1C33). AKR1C30 reduced only 17-keto-5ß-androstanes, whereas the other enzymes were active toward 3-, 17-, and 20-ketosteroids, and AKR1C33 further reduced 3-keto groups of bile acids and 7α-hydroxy-5ß-cholestanes. In addition, AKR1C30, AKR1C31, AKR1C32, and AKR1C33 were selectively inhibited by carbenoxolone, baccharin, phenolphthalein, and zearalenone, respectively. The mRNAs for the four enzymes were ubiquitously expressed in male rabbit tissues, in which highly expressed tissues were the brain, heart, liver, kidney, intestine, colon, and testis (for AKR1C30 and AKR1C31); brain, heart, liver, kidney, testis, lung, and adrenal gland (for AKR1C32); and liver and intestine (for AKR1C33). Thus, the four enzymes correspond to the multiple drug ketone reductases, and may function in the metabolisms of steroids, isatin and reactive carbonyl compounds, and bile acid synthesis.

Exposure to 9,10-phenanthrenequinone accelerates malignant progression of lung cancer cells through up-regulation of aldo-keto reductase 1B10.

Inhalation of 9,10-phenanthrenequinone (9,10-PQ), a major quinone in diesel exhaust, exerts fatal damage against a variety of cells involved in respiratory function. Here, we show that treatment with high concentrations of 9,10-PQ evokes apoptosis of lung cancer A549 cells through production of reactive oxygen species (ROS). In contrast, 9,10-PQ at its concentrations of 2 and 5 µM elevated the potentials for proliferation, invasion, metastasis and tumorigenesis, all of which were almost completely inhibited by addition of an antioxidant N-acetyl-l-cysteine, inferring a crucial role of ROS in the overgrowth and malignant progression of lung cancer cells. Comparison of mRNA expression levels of six aldo-keto reductases (AKRs) in the 9,10-PQ-treated cells advocated up-regulation of AKR1B10 as a major cause contributing to the lung cancer malignancy. In support of this, the elevation of invasive, metastatic and tumorigenic activities in the 9,10-PQ-treated cells was significantly abolished by the addition of a selective AKR1B10 inhibitor oleanolic acid. Intriguingly, zymographic and real-time PCR analyses revealed remarkable increases in secretion and expression, respectively, of matrix metalloproteinase 2 during the 9,10-PQ treatment, and suggested that the AKR1B10 up-regulation and resultant activation of mitogen-activated protein kinase cascade are predominant mechanisms underlying the metalloproteinase induction. In addition, HPLC analysis and cytochrome c reduction assay in in vitro 9,10-PQ reduction by AKR1B10 demonstrated that the enzyme catalyzes redox-cycling of this quinone, by which ROS are produced. Collectively, these results suggest that AKR1B10 is a key regulator involved in overgrowth and malignant progression of the lung cancer cells through ROS production due to 9,10-PQ redox-cycling.

Induction of aldo-keto reductases (AKR1C1 and AKR1C3) abolishes the efficacy of daunorubicin chemotherapy for leukemic U937 cells.

Continuous exposure to daunorubicin (DNR) confers resistance against the drug-elicited lethality of leukemic cells and then reduces the remission rate. However, the detailed mechanisms involved in resistance development of leukemic cells to DNR remain unclear. Upregulation of aldo-keto reductases (AKRs) in human leukemic U937 cells was evaluated by gene-specific PCR and western blot analyses, and the contribution of AKRs toward the DNR sensitivity was assessed using gene expression and RNA-interference techniques and specific inhibitors. In addition, DNR reduction and cell differentiation were analyzed by fluorescence high-performance liquid chromatography and flow cytometry, respectively. Treatment with high doses of DNR triggered apoptotic induction of U937 cells through the production of reactive oxygen species (ROS) and a ROS-dependent mechanism. In contrast, DNR, at its sublethal doses, induced the expression of AKR1C1 and AKR1C3, both of which reduced the DNR sensitivity of the cells. The enzymes did not interfere with the cell differentiation caused by DNR, whereas their upregulation facilitated reduction of the anticancer drug and a ROS-derived lipid aldehyde 4-hydroxy-2-nonenal. These results suggest crucial roles of AKR1C1 and AKR1C3 in the acquisition of DNR resistance of leukemic cells by metabolizing both DNR and cytotoxic aldehydes derived from ROS-linked lipid peroxidation.

Synthesis of non-prenyl analogues of baccharin as selective and potent inhibitors for aldo-keto reductase 1C3.

Inhibitors of a human member (AKR1C3) of the aldo-keto reductase superfamily are regarded as promising therapeutics for the treatment of prostatic and breast cancers. Baccharin [3-prenyl-4-(dihydrocinnamoyloxy)cinnamic acid], a component of propolis, was shown to be both potent (Ki 56 nM) and highly isoform-selective inhibitor of AKR1C3. In this study, a series of derivatives of baccharin were synthesized by replacing the 3-prenyl moiety with aryl and alkyl ether moieties, and their inhibitory activities for the enzyme were evaluated. Among them, two benzyl ether derivatives, 6m and 6n, showed an equivalent inhibitory potency to baccharin. The molecular docking of 6m in AKR1C3 has allowed the design and synthesis of (E)-3-{3-[(3-hydroxybenzyl)oxy]-4-[(3-phenylpropanoyl)oxy]phenyl}acrylic acid (14) with improved potency (Ki 6.4 nM) and selectivity comparable to baccharin. Additionally, 14 significantly decreased the cellular metabolism of androsterone and cytotoxic 4-oxo-2-nonenal by AKR1C3 at much lower concentrations than baccharin.

Probing AKR1C30 and AKR1C31 with site-directed mutagenesis: identifying the roles of residues 54 and 56 in the binding of substrates and inhibitors.

Five rabbit aldo-keto reductases (AKRs) that participate in the reduction of drug ketones and endogenous ketosteroids have recently been cloned and characterized. Among them, AKR1C30 and AKR1C31 show the highest amino acid sequence identity of 91%, but markedly differ in their substrate specificity and inhibitor sensitivity. AKR1C30 reduces two drugs (ketotifen and naloxone) and 17-keto-5ß-androstanes, whereas AKR1C31 does not reduce the two drugs, but is active towards loxoprofen and various 3/17/20-ketosteroids. In addition, AKR1C30 is selectively inhibited by carbenoxolone, valproic acid and phenobarbital. Residues A54 and R56 are located adjacent to the catalytic residue Y55 of AKR1C30. To clarify the determinants for the substrate specificity and inhibitor sensitivity of AKR1C30, we performed the mutagenesis of A54 and R56 to the corresponding residues (L and Q) of AKR1C31. The A54L mutation produced an enzyme that had almost the same substrate specificity as AKR1C31 and decreased the sensitivity to the inhibitors except for carbenoxolone. The R56Q mutation decreased the affinity for only carbenoxolone among the substrates and inhibitors. Thus, the difference in the properties between the two enzymes can be attributed to their residue difference at positions 54 and 56.

Characterization of rabbit morphine 6-dehydrogenase and two NAD(+)-dependent 3α(17ß)-hydroxysteroid dehydrogenases.

Mammalian morphine 6-dehydrogenase (M6DH)(1) converts morphine into a reactive electrophile, morphinone. M6DH belongs to the aldo-keto reductase (AKR) superfamily, but its endogenous substrates and entire amino acid sequence remain unknown. A recent rabbit genomic sequencing predicts three genes for novel AKRs (1C26, 1C27 and 1C28) that share >87% amino acid sequence identity and are similar to the partial sequence of rabbit liver M6DH. We isolated cDNAs for the three AKRs, and compared the properties of their recombinant enzymes. Like M6DH, only AKR1C26 that shares the highest sequence identity with hepatic M6DH oxidized morphine. The three AKRs showed NAD(+)-dependent dehydrogenase activity towards other non-steroidal alicyclic alcohols and 3α/17ß-hydroxy-C(18)/C(19)/C(21)-steroids, and their mRNAs were ubiquitously expressed in rabbit tissues. The kinetic constants for the substrates suggest that at least AKR1C26 and AKR1C28 act as NAD(+)-dependent 3α/17ß-hydroxysteroid dehydrogenases. AKR1C27 differed from AKR1C28 in its high K(m) values for the substrates and low sensitivity towards competitive inhibitors (ikarisoside A, hinokitiol, hexestrol and zearalenone), despite their 95% sequence identity. The site-directed mutagenesis of Tyr118 and Phe310 in AKR1C27 to the corresponding residues (Phe and Ile, respectively) in AKR1C28 produced an enzyme that was similar to AKR1C28, suggesting their key roles in ligand binding.

Synthesis and structure-activity relationship of 2-phenyliminochromene derivatives as inhibitors for aldo-keto reductase (AKR) 1B10.

Inhibitors of a human member (AKR1B10) of the aldo-keto reductase superfamily are regarded as promising therapeutics for the treatment of cancer. Recently, we have discovered (Z)-2-(4-methoxyphenylimino)-7-hydroxy-N-(pyridin-2-yl)-2H-chromene-3-carboxamide (1) as the potent competitive inhibitor using the virtual screening approach, and proposed its 4-methoxy group on the 2-phenylimino moiety as an essential structural prerequisite for the inhibition. In this study, 18 derivatives of 1 were synthesized and their inhibitory potency against AKR1B10 evaluated. Among them, 7-hydroxy-2-(4-methoxyphenylimino)-2H-chromene-3-carboxylic acid benzylamide (5n) was the most potent inhibitor showing a Ki value of 1.3nM. The structure-activity relationship of the derivatives indicated that the 7-hydroxyl group on the chromene ring, but not the 4-methoxy group, was absolutely required for inhibitory activity, The molecular docking of 5n in AKR1B10 and site-directed mutagenesis of the enzyme residues suggested that the hydrogen-bond interactions between the 7-hydroxyl group of 5n and the catalytic residues (Tyr49 and His111) of the enzyme, together with a π-stacking interaction of the benzylamide moiety of 5n with Trp220, are important for the potent inhibition.

Substrate specificity and inhibitor sensitivity of rabbit 20α-hydroxysteroid dehydrogenase.

In this study, we examined the substrate specificity and inhibitor sensitivity of rabbit 20α-hydroxysteroid dehydrogenase (AKR1C5), which plays a role in the termination of pregnancy by progesterone inactivation. AKR1C5 moderately reduced the 3-keto group of only 5α-dihydrosteroids with 17ß- or 20α/ß-hydroxy group among 3-ketosteroids. In contrast, the enzyme reversibly and efficiently catalyzed the reduction of various 17- and 20-ketosteroids, including estrogen precursors (dehydroepiandrosterone, estrone and 5α-androstan-3ß-ol-17-one) and tocolytic 5ß-pregnane-3,20-dione. In addition to the progesterone inactivation, the formation of estrogens and metabolism of the tocolytic steroid by AKR1C5 may be related to its role in rabbit parturition. AKR1C5 also reduced various non-steroidal carbonyl compounds, including isatin, an antagonist of the C-type natriuretic peptide receptor, and 4-oxo-2-nonenal, suggesting its roles in controlling the bioactive isatin and detoxification of cytotoxic aldehydes. AKR1C5 was potently and competitively inhibited by flavonoids such as kaempferol and quercetin, suggesting that its activity is affected by ingested flavonoids.

9,10-Phenanthrenequinone promotes secretion of pulmonary aldo-keto reductases with surfactant.

9,10-Phenanthrenequinone (9,10-PQ), a major quinone in diesel exhaust particles, induces apoptosis via the generation of reactive oxygen species (ROS) because of 9,10-PQ redox cycling. We have found that intratracheal infusion of 9,10-PQ facilitates the secretion of surfactant into rat alveolus. In the cultured rat lung, treatment with 9,10-PQ results in an increase in a lower-density surfactant by ROS generation through redox cycling of the quinone. The surfactant contains aldo-keto reductase (AKR) 1C15, which reduces 9,10-PQ and the enzyme level in the surfactant increases on treatment with 9,10-PQ suggesting an involvement of AKR1C15 in the redox cycling of the quinone. In six human cell types (A549, MKN45, Caco2, Hela, Molt4 and U937) only type II epithelial A549 cells secrete three human AKR1C subfamily members (AKR1C1, AKR1C2 and AKR1C3) with the surfactant into the medium; this secretion is highly increased by 9,10-PQ treatment. Using in vitro enzyme inhibition analysis, we have identified AKR1C3 as the most abundantly secreted AKR1C member. The AKR1C enzymes in the medium efficiently reduce 9,10-PQ and initiate its redox cycling accompanied by ROS production. The exposure of A549 cells to 9,10-PQ provokes viability loss, which is significantly protected by the addition of the AKR1C3 inhibitor and antioxidant enzyme and by the removal of the surfactants from the culture medium. Thus, the AKR1C enzymes secreted in pulmonary surfactants probably participate in the toxic mechanism triggered by 9,10-PQ.

Characterization of rabbit aldose reductase-like protein with 3ß-hydroxysteroid dehydrogenase activity.

In this study, we isolated the cDNA for a rabbit aldose reductase-like protein that shared an 86% sequence identity to human aldo-keto reductase (AKR)(1) 1B10 and has been assigned as AKR1B19 in the AKR superfamily. The purified recombinant AKR1B19 was similar to AKR1B10 and rabbit aldose reductase (AKR1B2) in the substrate specificity for various aldehydes and α-dicarbonyl compounds. In contrast to AKR1B10 and AKR1B2, AKR1B19 efficiently reduced 3-keto-5α/ß-dihydro-C19/C21/C24-steroids into the corresponding 3ß-hydroxysteroids, showing K(m) of 1.3-9.1 µM and k(cat) of 1.1-7.6 min(-1). The stereospecific reduction was also observed in the metabolism of 5α- and 5ß-dihydrotestosterones in AKR1B19-overexpressing cells. The mRNA for AKR1B19 was ubiquitously expressed in rabbit tissues, and the enzyme was co-purified with 3ß-hydroxysteroid dehydrogenase activity from the lung. Thus, AKR1B19 may function as a 3-ketoreductase, as well as a defense system against cytotoxic carbonyl compounds in rabbit tissues. The molecular determinants for the unique 3-ketoreductase activity were investigated by replacement of Phe303 and Met304 in AKR1B19 with Gln and Ser, respectively, in AKR1B10. Single and double mutations (F303Q, M304S and F303Q/M304S) significantly impaired this activity, suggesting the two residues play critical roles in recognition of the steroidal substrate.

GABA production by glutamic acid decarboxylase is regulated by a dynamic catalytic loop.

Gamma-aminobutyric acid (GABA) is synthesized by two isoforms of the pyridoxal 5'-phosphate-dependent enzyme glutamic acid decarboxylase (GAD65 and GAD67). GAD67 is constitutively active and is responsible for basal GABA production. In contrast, GAD65, an autoantigen in type I diabetes, is transiently activated in response to the demand for extra GABA in neurotransmission, and cycles between an active holo form and an inactive apo form. We have determined the crystal structures of N-terminal truncations of both GAD isoforms. The structure of GAD67 shows a tethered loop covering the active site, providing a catalytic environment that sustains GABA production. In contrast, the same catalytic loop is inherently mobile in GAD65. Kinetic studies suggest that mobility in the catalytic loop promotes a side reaction that results in cofactor release and GAD65 autoinactivation. These data reveal the molecular basis for regulation of GABA homeostasis.

Structure of the His269Arg mutant of the rat aldose reductase-like protein AKR1B14 complexed with NADPH.

Rat aldose reductase-like protein (AKR1B14) is an orthologue of mouse vas deferens protein (AKR1B7) and plays roles in the detoxification of reactive aldehydes and synthesis of prostaglandin F(2α). Here, the 1.87 Å resolution crystal structure of the His269Arg mutant of AKR1B14 complexed with NADPH is described and shows that the negatively charged 2'-phosphate group of the coenzyme forms an ionic interaction with the positively charged guanidinium group of Arg269 that is also observed in the human aldose reductase (AKR1B1) structure. Previous experiments on the site-directed mutagenesis of His269 to Arg, Phe and Met revealed fourfold, sevenfold and 127-fold increases in the K(m) for NADPH, respectively, which are in agreement with the present molecular-modelling and X-ray crystallographic studies. This is the first tertiary structure of a mutant form of this AKR1B7 orthologue to be reported in order to investigate the structure-function relationship of the nonconserved His269 and its role in coenzyme binding.

Selective inhibition of human type-5 17ß-hydroxysteroid dehydrogenase (AKR1C3) by baccharin, a component of Brazilian propolis.

The human aldo-keto reductase (AKR) 1C3, also known as type-5 17ß-hydroxysteroid dehydrogenase and prostaglandin F synthase, has been suggested as a therapeutic target in the treatment of prostate and breast cancers. In this study, AKR1C3 inhibition was examined by Brazilian propolis-derived cinnamic acid derivatives that show potential antitumor activity, and it was found that baccharin (1) is a potent competitive inhibitor (K(i) 56 nM) with high selectivity, showing no significant inhibition toward other AKR1C isoforms (AKR1C1, AKR1C2, and AKR1C4). Molecular docking and site-directed mutagenesis studies suggested that the nonconserved residues Ser118, Met120, and Phe311 in AKR1C3 are important for determining the inhibitory potency and selectivity of 1. The AKR1C3-mediated metabolism of 17-ketosteroid and farnesal in cancer cells was inhibited by 1, which was effective from 0.2 µM with an IC(50) value of about 30 µM. Additionally, 1 suppressed the proliferation of PC3 prostatic cancer cells stimulated by AKR1C3 overexpression. This study is the first demonstration that 1 is a highly selective inhibitor of AKR1C3.