The Influence of Some Nonsteroidal Anti-inflammatory Drugs on Metabolic Enzymes of Aldose Reductase, Sorbitol Dehydrogenase, and α-Glycosidase: a Perspective for Metabolic Disorders.

Pain, as a sensible alarm signal of living organisms to avoid tissue damage, is a common and debilitating consequence of a lot of disorders and diseases. The management of chronic pain is particularly challenging. For pain treatment, many analgesic drugs are used for their therapeutic effects. In this study, some nonsteroidal anti-inflammatory drugs including etofenamate, meloxicam, diclofenac, and tenoxicam were tested against α-glycosidase from Saccharomyces cerevisiae, sorbitol dehydrogenase (SDH), and aldose reductase (AR) enzymes from sheep liver. Nonsteroidal anti-inflammatory drugs demonstrated useful inhibition properties against α-glycosidase, AR, and SDH enzymes. Ki values were found in the range of 11.93 ± 3.77-364.88 ± 40.01 µM for α-glycosidase, 3.36 ± 1.08µM-17.68 ± 3.39 mM for AR, and 1.68 ± 0.02 µM-30.98 ± 14.31 mM for SDH. They can be selective drugs as antidiabetic agents, because of their inhibitory properties against SDH, α-glycosidase, and AR enzymes.

The interactions of cephalosporins on polyol pathway enzymes from sheep kidney.

CONTEXT: Cephalosporins are derived from the fungus Acremonium. Due to their strong bactericidal ability, these drugs have to a wide usage in medicine. OBJECTIVE: An investigation of the effects on sheep renal aldose reductase (AR) and sorbitol dehydrogenase (SDH) of cefoperazone, cefazolin, cefuroxime, ceftazidime and ceftriaxone as cephalosporin drugs was carried out in the present study. METHODS: AR and SDH were purified from sheep kidney by ion exchange, gel filtration and affinity methods with approximately 219- and 484-fold, respectively. Some kinetic properties of the enzymes were determined such as optimal pH, optimal ionic strength, optimal temperature, stable pH, Km and Vmax. IC50 values of the drugs were found for each enzyme. RESULTS: While the AR was inhibited by all drugs, SDH enzyme was inhibited by only CXM (IC50 8.10 mM). Interestingly, CZO activated SDH enzyme. This result was evaluated as important for the flow of the polyol reactions. Ki values and inhibition types were determined for AR. However, these values could not have determined for SDH, due to insufficient inhibition. CONCLUSIONS: From these results, it was concluded that cephalosporins may have an important effect on flow of the polyol metabolism.

A highly efficient sorbitol dehydrogenase from Gluconobacter oxydans G624 and improvement of its stability through immobilization.

A sorbitol dehydrogenase (GoSLDH) from Gluconobacter oxydans G624 (G. oxydans G624) was expressed in Escherichia coli BL21(DE3)-CodonPlus RIL. The complete 1455-bp codon-optimized gene was amplified, expressed, and thoroughly characterized for the first time. GoSLDH exhibited Km and kcat values of 38.9 mM and 3820 s(-1) toward L-sorbitol, respectively. The enzyme exhibited high preference for NADP(+) (vs. only 2.5% relative activity with NAD(+)). GoSLDH sequencing, structure analyses, and biochemical studies, suggested that it belongs to the NADP(+)-dependent polyol-specific long-chain sorbitol dehydrogenase family. GoSLDH is the first fully characterized SLDH to date, and it is distinguished from other L-sorbose-producing enzymes by its high activity and substrate specificity. Isothermal titration calorimetry showed that the protein binds more strongly to D-sorbitol than other L-sorbose-producing enzymes, and substrate docking analysis confirmed a higher turnover rate. The high oxidation potential of GoSLDH for D-sorbitol was confirmed by cyclovoltametric analysis. Further, stability of GoSLDH significantly improved (up to 13.6-fold) after cross-linking of immobilized enzyme on silica nanoparticles and retained 62.8% residual activity after 10 cycles of reuse. Therefore, immobilized GoSLDH may be useful for L-sorbose production from D-sorbitol.

Functional assignment of gene AAC16202.1 from Rhodobacter capsulatus SB1003: new insights into the bacterial SDR sorbitol dehydrogenases family.

Short-chain dehydrogenases/reductases (SDR) constitute one of the largest enzyme superfamilies with over 60,000 non-redundant sequences in the database, many of which need a correct functional assignment. Among them, the gene AAC16202.1 (NCBI) from Rhodobacter capsulatus SB1003 has been assigned in Uniprot both as a sorbitol dehydrogenase (#D5AUY1) and, as an N-acetyl-d-mannosamine dehydrogenase (#O66112), both enzymes being of biotechnological interest. When the gene was overexpressed in Escherichia coli Rosetta (DE3)pLys, the purified enzyme was not active toward N-acetyl-d-mannosamine, whereas it was active toward d-sorbitol and d-fructose. However, the relative activities toward xylitol and l-iditol (0.45 and 6.9%, respectively) were low compared with that toward d-sorbitol. Thus, the enzyme could be considered sorbitol dehydrogenase (SDH) with very low activity toward xylitol, which could increase its biotechnological interest for determining sorbitol without the unspecific cross-determination of added xylitol in food and pharma compositions. The tetrameric enzyme (120 kDa) showed similar catalytic efficiency (2.2 × 10(3) M(-1) s(-1)) to other sorbitol dehydrogenases for d-sorbitol, with an optimum pH of 9.0 and an optimum temperature of 37 °C. The enzyme was also more thermostable than other reported SDH, ammonium sulfate being the best stabilizer in this respect, increasing the melting temperature (T(m)) up to 52.9 °C. The enzyme can also be considered as a new member of the Zn(2+) independent SDH family since no effect on activity was detected in the presence of divalent cations or chelating agents. Finally, its in silico analysis enabled the specific conserved sequence blocks that are the fingerprints of bacterial sorbitol dehydrogenases and mainly located at C-terminal of the protein, to be determined for the first time. This knowledge will facilitate future data curation of present databases and a better functional assignment of newly described sequences.

Effects of some anti-neoplastic drugs on sheep liver sorbitol dehydrogenase.

Stress is an important factor for many diseases in living metabolisms. The mini pathway named as polyol is a critical junction for stress factors. This pathway has two enzymes: aldose reductase (AR) and sorbitol dehydrogenase (SDH). It is linked with some diseases such as diabetes mellitus and some cancer types. In particular, SDH is very sensitive and unstable in in vitro conditions. In this study, SDH was purified by using simple and rapid chromatographic methods such as DEAE-Sephadex and CM-Sephadex C-50 columns. Subunit and active form molecular weights were found as 39.8 kDa and 150 kDa, respectively. The in vitro effects of some antineoplastic drugs were investigated. IC(50) values were 0.025, 0.081, 0.291, 1.62, 4.86, 6.54 mM for dacarbazine, methotrexate, epirubicin hydrochloride, calcium folinate, gemcitabine hydrochloride, oxaliplatin, respectively. From these results, dacarbazine was lowest IC(50) value and it is the strongest inhibitor for liver SDH enzyme activity compared to the other drugs.

Reversible phosphorylation regulation of NADPH-linked polyol dehydrogenase in the freeze-avoiding gall moth, Epiblema scudderiana: role in glycerol metabolism.

Larvae of the goldenrod gall moth, Epiblema scudderiana, use a freeze avoidance strategy of cold hardiness to survive the winter. A key metabolic adaption that supports subzero survival is the accumulation of large amounts of glycerol as a colligative antifreeze. Production of glycerol relies on polyol dehydrogenase (PDH) which catalyzes the NADPH-dependent conversion of glyceraldehyde into glycerol. Kinetic analysis of PDH from E. scudderiana revealed significant changes in properties as a result of subzero temperature acclimation; the K(m) for glyceraldehyde in 5°C-acclimated larvae was 7.0 mM and doubled in - 15°C-exposed larvae. This change suggested that PDH is regulated by a state-dependent covalent modification. Indeed, high and low K(m) forms could be interconverted by incubating larval extracts in vitro under conditions that stimulated either endogenous protein kinases or protein phosphatases. Protein kinase incubations doubled the K(m) glyceraldehyde of the 5°C enzyme, whereas protein phosphatase incubations decreased the K(m) of the - 15°C enzyme by about 50%. PDH was purified by ion exchange and affinity chromatography steps and then subjected to electrophoresis. Staining with ProQ Diamond phosphoprotein stain showed a much higher phosphate content of PDH from - 15°C-acclimated larvae, a result that was further confirmed by immunoblotting that showed a much greater phosphoserine content on the - 15°C enzyme. These experiments established that PDH is regulated by state-dependent reversible phosphorylation in E. scudderiana and suggest that this regulatory mechanism makes a significant contribution to controlling the synthesis, maintenance, and degradation of glycerol pools over the winter months.

Cloning of cDNAs encoding sorbitol dehydrogenase-2a and b, enzymatic characterization, and up-regulated expression of the genes in Bombyx mori diapause eggs exposed to 5 °C.

We previously cloned a cDNA for sorbitol dehydrogenase (SDH1) from Bombyx mori. In the present study we cloned two additional cDNAs encoding SDHs (designated as SDH2a and SDH2b). The amino acid sequences of SDH2ab were almost the same and had higher similarity to the SDHs of other organisms than to B. mori SDH1. The SDH2ab and SDH1genes were located in tandem within about 40 kbp on chromosome 21. SDH2ab mRNAs increased after exposing diapause eggs to 5 °C for 40 days, beginning at 2 days post-oviposition, to break diapause. However, they were at very low levels in diapausing eggs incubated at 25 °C continuously from oviposition. These changes in expression pattern of SDH2ab mRNA were almost the same as that of SDH1 mRNA. To understand whether SDH1 and SDH2 were responsible for the SDH activity seen in diapause eggs exposed to 5 °C for more than 60 days, we expressed a His-tagged SDH2a fusion protein in Escherichia coli and examined its enzymatic parameters. The maximum activity of SDH2a observed at pH 8.4∼9.0, and the Km value for sorbitol was 12.6 mM, similar to the kinetic properties of other SDHs. Due to the significantly higher similarity between SDH2a and b, they were thought to have similar kinetic properties. Therefore, we purified SDH from B. mori diapause-terminated eggs exposed to 5 °C for 300 days which showed higher SDH activity using two-step affinity chromatography. The highly purified SDH showed a higher Km value (125 mM) for sorbitol, being similar to the value (136 mM) determined previously from Eadie-Hofstee plots using egg crude extract as an enzyme source; additionally, the plots showed one slope indicating one Km value. Moreover, in silico analysis indicated that no SDH genes other than SDH1 and 2ab are present in B. mori genomic DNA. These results suggest that SDH1 activity may be responsible for the majority of the increased SDH activity seen in diapause eggs after acclimation to 5 °C rather than SDH2ab. Further, the relative sequence divergence among these genes is consistent with the idea/hypothesis that the original SDH gene was first duplicated into SDH1 and SDH2, and then SDH2 was duplicated into the SDH2a and SDH2b genes.

Enzyme separation and isozyme heterogeneity analysis using non-denaturing two-dimensional electrophoresis.

Carboxylesterase and sorbitol dehydrogenase are separated by non-denaturing two-dimensional electrophoresis (2-DE) of isoelectric focusing separation using 5% carrier ampholyte (pH 6-8) and 1.25% carrier ampholyte (pH 3-10) and size separation. Furthermore, activities of sorbitol, malate and lactate dehydrogenases are sequentially examined when the enzymes are separated by 2-DE and are sequentially reacted to sorbitol, malic and lactic acid, respectively, in the presence of nicotinamide adenine dinucleotide, nitro blue tetrazolium and phenazine methosulphate. Several kinds of enzymes including lactate dehydrogenize isozymes can be simultaneously separated using 2-DE. Furthermore, the binding differences between lactate dehydrogenase isozymes and concanavalin A (con A) can be examined using a combination of 2-DE and non-denaturing stacking gel electrophoresis. The results of this study indicate that non-denaturing 2-DE can be applied to both enzyme separation and isozyme heterogeneity analysis.

Ubiquitous distribution and different subcellular localization of sorbitol dehydrogenase in fruit and leaf of apple.

NAD(+)-dependent sorbitol dehydrogenase (NAD-SDH, EC 1.1.1.14), a key enzyme in sorbitol metabolism, plays an important role in regulating sink strength and determining the quality of apple fruit. Understanding the tissue and subcellular localization of NAD-SDH is helpful for understanding sorbitol metabolism in the apple. In this study, two NAD-SDH cDNA sequences were isolated from apple fruits (Malus domestica Borkh cv. Starkrimson) and named MdSDH5 and MdSDH6. Immunohistochemical analysis revealed that NAD-SDH is distributed in both the flesh and the vascular tissue of the fruit, and the vascular tissue and mesophyll tissue in the young and old leaves, indicating that it is a ubiquitous protein expressed in both sink and source organs. Immunogold electron microscopy analysis demonstrated that NAD-SDH is localized mainly in the cytoplasm and chloroplast of the fruit and leaves. The chloroplast localization of NAD-SDH was confirmed by the transient expression of MdSDH5-GFP and MdSDH6-GFP in the mesophyll protoplast of Arabidopsis. NAD-SDH was also found in electron opaque deposits of vacuoles in young and mature leaves. These data show that NAD-SDH has different subcellular localizations in fruit and leaves, indicating that it might play a different role in sorbitol metabolism in different tissues of apple.

Membrane-bound pyrroloquinoline quinone-dependent dehydrogenase in Gluconobacter oxydans M5, responsible for production of 6-(2-hydroxyethyl) amino-6-deoxy-L-sorbose.

A membrane-bound protein purified from Gluconobacter oxydans M5 was confirmed to be a pyrroloquinoline quinone-dependent D-sorbitol dehydrogenase. Gene disruption and complementation experiments demonstrated that this enzyme is responsible for the oxidation of 1-(2-hydroxyethyl) amino-1-deoxy-D-sorbitol (1NSL) to 6-(2-hydroxyethyl) amino-6-deoxy-L-sorbose (6NSE), which is the precursor of an antidiabetic drug, miglitol.

Purification and properties of NAD(P)-independent polyol dehydrogenase complex from the plasma membrane of Gluconobacter oxydans.

Gluconobacter oxydans rapidly oxidizes many different polyhydroxy alcohols (polyols). Polyol oxidations are catalyzed by constitutively synthesized membrane-bound dehydrogenases directly linked to the electron transport chain. A polyol-oxidizing enzyme was isolated from the membranes of G. oxydans and tested for its ability to oxidize various substrates. The enzyme was composed of three subunits: a 67 kDa catalytic unit, a 46 kDa c-type cytochrome, and a 15 kDa subunit. The enzyme oxidized compounds containing three or more hydroxyl groups but did not oxidize mono-, di-, or cyclic alcohols; aldehydes; carboxylic acids; or mono- or di-saccharides. Therefore, we propose this enzyme be considered a polyol dehydrogenase.

Discovery of potential sorbitol dehydrogenase inhibitors from virtual screening.

Sorbitol dehydrogenase (SDH) is the second enzyme in the polyol pathway of glucose metabolism and is a possible target for the treatment of the complications of diabetes. In this study the molecular modelling program DOCK was used to analyse 249,071 compounds from the National Cancer Institute Database and predict those with high affinity for SDH. From a total of 21 tested the 7 compounds including flavin adenine dinucleotide disodium hydrate, (+)-Amethopterin, 3-hydroxy-2-napthoic(2-hydroxybenzylidene) hydrazide, folic acid, N-2,4-dinitrophenyl-L-cysteic acid, Vanillin azine and 1H-indole-2,3-dione,5-bromo-6-nitro-1-(2,3,4-tri-O-acetyl-alpha-L-arabinopyranosyl)-(9Cl), were shown to inhibit SDH and displayed IC50 values of 0.192 microM, 1.1 microM, 1.2 microM, 4.5 microM, 5.3 microM, 7 microM and 28 microM, respectively. These compounds may aid the design of pharmaceutical agents for the treatment of diabetes complications.

Expression, purification and preliminary crystallographic analysis of human sorbitol dehydrogenase.

Human sorbitol dehydrogenase (SDH) was expressed in Escherichia coli BL21 cells and purified using ammonium sulfate precipitation and anion-exchange and dye-affinity chromatography. Purified SDH was crystallized from polyethylene glycol solutions using the hanging-drop vapour-diffusion method. X-ray data were collected to 2.75 A resolution. The crystals belong to the monoclinic C2 space group, with unit-cell parameters a = 145.9, b = 52.3, c = 169.0 A, beta = 101.8 degrees. This is the first crystallization report of human sorbitol dehydrogenase.

Purification and properties of membrane-bound D-sorbitol dehydrogenase from Gluconobacter suboxydans IFO 3255.

D-Sorbitol dehydrogenase was solubilized from the membrane fraction of Gluconobacter suboxydans IFO 3255 with Triton X-100 in the presence of D-sorbitol. Purification of the enzyme was done by fractionation with column chromatographies of DEAE-Cellulose, DEAE-Sepharose, hydroxylapatite, and Sephacryl HR300 in the presence of Triton X-100. The molecular mass of the enzyme was 800 kDa, consisting of homologous subunits of 80 kDa. The optimum pH of the enzyme activity was 6.0, and the optimum temperature was 30 degrees C. Western blot analysis suggested the occurrence of the enzyme in all the Gluconobacter strains tested.

Purification and characterization of a NAD+-dependent sorbitol dehydrogenase from Japanese pear fruit.

NAD+-dependent sorbitol dehydrogenase NAD-SDH, EC 1.1.1.14) from Japanese pear fruit was purified to apparent homogeneity (single band by SDS-PAGE with silver staining), and had a specific activity of 916.7 nKatal/mg protein. The molecular of the native enzyme was calculated to be 160 kDa by gel filtration, whereas SDS-PAGE gave a subunit size of 40 kDa, indicating that the native enzyme is a homotetramer. The protein immunologically reacted with an antibody raised in rabbit against the fusion protein expressed in E. coli harboring an apple NAD-SDH cDNA. The Km, values for sorbitol and fructose were 96.4+/-8.60 and 4239+/-33.5 mM, respectively, and optimum pH for sorbitol oxidation was 9.0 and 7.0 for fructose reduction. Pear NAD-SDH had a very narrow substrate specificity, that is, sorbitol, L-iditol, xylitol and L-threitol were oxidized but not any of the other alcohols tested. These data suggest the structural importance of an S configuration at C-2 and an R configuration at C-4 in the substrate(s). Its enzymatic activity was strongly inhibited both by heavy metal ions such as mercury, and by thiol compounds, such as L-cysteine. However, the addition of zinc ion reversed the enzyme inactivation caused by addition of L-cysteine.

Sorbitol dehydrogenase from bovine lens: purification and properties.

Bovine lens sorbitol dehydrogenase (L-iditol:NAD+ 2-oxidoreductase, EC 1.1.1.14) (SDH) was purified to electrophoretic homogeneity (51 U/mg of protein) and characterized for both kinetic and some structural properties. The enzyme proves to be a homotetramer of 156 kDa containing one equivalent of zinc ion per subunit. Metal chelators such as EDTA and 1,10-phenanthroline determine a loss of enzyme activity which can be specifically recovered by addition of either zinc or manganese ions. Inactivation induced not only by metal chelators but also by thiol reagents is effectively prevented by the pyridine cofactor. Bovine lens SDH is active on polyalcohols and keto-sugars with more than three carbon atoms, and also requires special steric constraints for substrate recognition. Of the polyols, xylitol is the most effective substrate (kcat/KM of 8.1 s-1 mM-1), followed by sorbitol (kcat/KM of 1.59 s-1 mM-1); fructose, the most effective carbonyl substrate, displays a kcat/KM of only 0.9 s-1 mM-1. Analysis at the steady state of initial velocities as a function of the concentration of different substrates and cofactors and studies of product inhibition indicate for both fructose reduction and sorbitol oxidation a Theorell and Chance-type kinetic mechanism of action.

Glycation and inactivation of sorbitol dehydrogenase in normal and diabetic rats.

Sorbitol dehydrogenase (SDH) is involved in the polyol pathway, which plays an important role in the pathogenesis of diabetic complications. We have measured the tissue distributions of SDH mRNA, both the immunoreactive enzyme levels and the enzyme activity. SDH mRNA was especially abundant in liver, kidney and testis. Both the activity and enzyme content are high in liver and kidney but not in testis. The discrepancy between mRNA and immunoreactive enzyme levels and the activity of SDH observed in testis was also seen in livers of streptozotocin-induced diabetic rats. SDH was found to exist in both glycated and non-glycated forms, with larger amounts of the glycated protein in the diabetic liver. Moreover, after incubation of purified enzyme with glucose or fructose, its activity was markedly decreased. These results indicate that glycation causes a decrease in SDH activity in liver under diabetic conditions. The same post-transcriptional event might occur to decrease the activity of SDH in testis in normal animals.

Polyol metabolism of Rhodobacter sphaeroides: biochemical characterization of a short-chain sorbitol dehydrogenase.

A sorbitol dehydrogenase (SDH; L-iditol:NAD+ 2-oxidoreductase; EC 1.1.1.14) was isolated from the phototrophic bacterium Rhodobacter sphaeroides strain M22, a transposon mutant of R. sphaeroides Si4 with the transposon inserted in the mannitol dehydrogenase (MDH) gene. SDH was purified 470-fold to apparent homogeneity by ammonium sulfate precipitation, chromatography on Phenyl-Sepharose, Q-Sepharose and Matrex Gel Red-A, and by gel filtration on Superdex 200. The relative molecular mass (M(r)) of the native SDH was 61000 as calculated from its Stokes' radius (rs = 3.5 nm) and sedimentation coefficient (S20,w = 4.23S). SDS-PAGE resulted in one single band representing a polypeptide with a M(r) of 29,000, indicating that the native protein is a dimer. The isoelectric point of SDH was determined to be pH 4.8. The enzyme was specific for NAD+ and catalysed the oxidation of D-glucitol (sorbitol) to D-fructose, galactitol to D-tagatose and of L-iditol. The apparent Km values were NAD+, 0.06 mM; D-glucitol, 6.2 mM; galactitol, 1.5 mM; NADH, 0.13 mM; D-fructose, 160 mM; and D-tagatose, 13 mM. The pH-optimum of substrate oxidation was 11.0 and that of substrate reduction 6.0-7.2. It was demonstrated that SDH is expressed in the wild-type strain R. sphaeroides Si4 together with MDH during growth on D-glucitol. Forty-four amino acids of the SDH N terminus were sequenced. This sequence exhibited 45-55% identity to the N-terminal sequence of 10 enzymes belonging to the short-chain alcohol dehydrogenase family.

The human sorbitol dehydrogenase gene: cDNA cloning, sequence determination, and mapping by fluorescence in situ hybridization.

The cDNA for human sorbitol dehydrogenase (SORD) has been cloned and sequenced. It translates into a peptide of 356 amino acid residues, one more than the sequence previously reported from peptide analysis. An extra alanine was found at the acetyl-blocked N-terminal, between positions 1 and 4. This matches the rat cDNA, which also has 356 amino acids, with an extra proline at position 3. Four other mis-matches were also observed, but these are all amino acid substitutions that occur outside proposed functionally important regions. Further work must be performed to determine whether these discrepancies represent polymorphic forms of the enzyme. The SORD gene was mapped by fluorescence in situ hybridization and found to occupy a single site on chromosome 15q15, indicating that it is a single-copy gene. This was confirmed by Southern blot hybridization. SORD is thought to be involved in the etiology of diabetic complications, and its deficiency has been linked to congenital cataracts. The cloned gene could be used as a probe to study the role of this enzyme in the pathogenesis of these diseases.

Sorbitol dehydrogenase from Bacillus subtilis. Purification, characterization, and gene cloning.

Cloning of the sorbitol dehydrogenase gene (gutB) from Bacillus subtilis offers an excellent system for studying zinc binding, substrate specificity, and catalytic mechanism of this enzyme through protein engineering. As a first step to clone gutB, B. subtilis sorbitol dehydrogenase has been purified to homogeneity and characterized. It is a tetrameric enzyme with a molecular mass of 38 kDa for each subunit. Atomic absorption analysis shows the presence of 1 mol of zinc atom/subunit. Substrate specificity and stereospecificity of the enzyme toward C-2 and C-4 of hexitols were established. Sequence of the first 31 amino acids was determined, and a set of oligonucleotide probes was designed for gene cloning. A positive clone carrying a 5-kilobase pair HindIII insert was isolated and sequenced. Sequence alignment indicated that the deduced amino acid sequence of B. subtilis sorbitol dehydrogenase shows 36% identity in sequence with the liver sorbitol dehydrogenase from sheep, rat, and human. In reference to the sequence of alcohol dehydrogenase, two potential zinc binding sites were identified. Sequence information related to the structure-function relationships of the enzyme is discussed.