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Yuguo Zheng - One of the best experts on this subject based on the ideXlab platform.
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combinational expression of d Sorbitol Dehydrogenase and pyrroloquinoline quinone increases 6 n hydroxyethyl amino 6 deoxy α l sorbofuranose production by gluconobacter oxydans through cofactor manipulation
Enzyme and Microbial Technology, 2020Co-Authors: Dong Liu, Yuguo ZhengAbstract:Abstract 6-(N-hydroxyethyl)-amino-6-deoxy- l -sorbofuranose (6NSL), a key precursor in the synthesis of miglitol, is produced from N-2-hydroxyethyl-glucamine (NHEG) by the regioselective oxidation of Gluconobacter oxydans. The limitation of PQQ biosynthesis became a bottleneck for improvement of PQQ-dependent D -Sorbitol Dehydrogenase (mSLDH) activity. Five expression plasmids were constructed for the co-expression of the pqqABCDE gene cluster and the tldD gene on the basis of pBBR1-gHp0169-sldAB in G. oxydans to increase the biosynthesis of PQQ. The G. oxydans/pGA004, in which pqqABCDE and tldD were expressed as a cluster under the control of gHp0169 promoter, showed the optimal performance. The intracellular PQQ concentration and specific activity of mSLDH in cells increased by 79.3 % and 53.7 %, respectively, compared to that in G. oxydans/pBBR-sldAB. Then, the repeated batch biotransformation of NHEG to 6NSL by G. oxydans/pGA004 was carried out. Up to 75.0 ± 3.0 g/L of 6NSL production with 94.5 ± 3.6 % of average conversion rate of NHEG to 6NSL was achieved after four cycles of run. These results indicated that G. oxydans/pGA004 with high productivity had great potential for 6NSL production in industrial bioprocess.
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breeding of gluconobacter oxydans with high pqq dependent d Sorbitol Dehydrogenase for improvement of 6 n hydroxyethyl amino 6 deoxy α l sorbofuranose production
Biochemical Engineering Journal, 2020Co-Authors: Dong Liu, Yuguo ZhengAbstract:Abstract 6-(N-hydroxyethyl)-amino-6-deoxy-L-sorbofuranose (6NSL), a key intermediate in the synthesis of miglitol, was produced from N-2-hydroxyethyl glucamine (NHEG) by biotransformation with whole cells of Gluconobacter oxydans. The main troubles in 6NSL production were the low activity of PQQ-dependent D-Sorbitol Dehydrogenase (mSLDH) in G. oxydans and the high cost of cell preparation. To solve these problems, a combined mutagenesis of 60Co-γ irradiation and microwave treatment with a high-throughput screening method by cultivation in a 96-well microtiter plate was conducted. After several cycles of mutagenesis, a stable mutant H-8 with high mSLDH activity was obtained, and the cell biomass increased by 11.6% when cultivated in a 5 L bioreactor. The transcription levels of the mSLDH subunit sldA and sldB in G. oxydans H-8 increased by 1.4- and 2.0-fold, respectively. Meanwhile, the intracellular PQQ concentration in G. oxydans H-8 was 16.1% higher than that of the parent strain, and qRT-PCR analysis showed that the genes pqqB and pqqC played an important role in PQQ biosynthesis (transcription levels increased by 2.4- and 1.8-fold, respectively). Relying on the advantages of the above, G. oxydans H-8 could produce 64.3 ± 2.2 g/L 6NSL after 36 h of bioconversion with resting cells, showing a 33.7% increase in the product yield.
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glutamate addition improves the activity of membrane bound Sorbitol Dehydrogenase in a pyrroloquinoline quinone dependent manner a feasible strategy for the cost effective fermentation of gluconobacter oxydans
Process Biochemistry, 2019Co-Authors: Liang Chen, Xinqiang Sun, Yuguo ZhengAbstract:Abstract Existing industrial fermentations for Gluconobacter oxydans require a rich nitrogen source, which results in a relatively high production cost. In present study, amino acids utilization from yeast extract (YE) (20 g·L−1) was investigated, revealing that glutamate is disproportionately utilized, and consequently becomes a rate-limiting factor for the activity of membrane-bound Sorbitol Dehydrogenase (mSLDH). Additional supplementation of glutamate (0.1%) improved the abundance of coenzyme pyrroloquinoline quinone (PQQ) by 1.51-fold. Meanwhile, enzyme activity of mSLDH was improved by 1.21-fold, which was mainly dependent on the induction of PQQ. Partial substitution of YE (15 g·L−1) with glutamate (2 g·L−1) as nitrogen source was tested in G. oxydans fermentations and the thus-obtained resting-cells exhibited an high mSLDH activity towards the miglitol precursor N-2-hydroxyethyl-glucamine with an increased productivity (59.2 vs. 42.1 mg· (g DCW)-1 h-1) and an equally high yield (81.56% vs. 80.35%) compared with cells fermented with 25 g·L−1 YE. Taken together, the results provide a feasible strategy for economical fermentation of G. oxydans in achieving high mSLDH activity for biotransformation applications in large-scale.
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synergistic improvement of pqq dependent d Sorbitol Dehydrogenase activity from gluconobacter oxydans for the biosynthesis of miglitol precursor 6 n hydroxyethyl amino 6 deoxy α l sorbofuranose
Journal of Biotechnology, 2019Co-Authors: Yu Panhong, Liang Chen, Xinqiang Sun, Yuguo ZhengAbstract:Abstract 6-(N-hydroxyethyl) amino-6-deoxy- l -sorbofuranose (6NSL) is the direct precursor of miglitol for diabetes therapy. The regio- and stereo-selective dehydrogenation offered by the membrane-bound d -Sorbitol Dehydrogenase (mSLDH) from Gluconobacter oxydans provides an elegant enzymatic method for 6NSL production. In this study, two subunits sldA and sldB of mSLDH were introduced into G. oxydans ZJB-605, and the specific enzyme activity of mSLDH towards NHEG was enhanced by 2.15-fold. However, the endogenous PQQ level was dramatically reduced in the recombinant strain and became a bottleneck to support the holo-enzyme activity. A combined supplementation of four amino acids (Glu, Ile, Ser, Arg) involved in biosynthesis of PQQ in conventional media effectively increased extracellular accumulation of PQQ by 1.49-fold, which further enhanced mSLDH activity by 1.33-fold. The synergic improvement of mSLDH activity provided in this study supports the superior high dehydrogenate activity towards substrate N-2-hydroxyethyl-glucamine, 184.28 g·L−1 of 6NSL was produced after a repeated bioconversion process catalyzed by the resting cells of G. oxydans/pBB-sldAB, all of which presenting a great potential of their industrial application in 6NSL biosynthesis.
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biosynthesis of miglitol intermediate 6 n hydroxyethyl amino 6 deoxy α l sorbofuranose by an improved d Sorbitol Dehydrogenase from gluconobacter oxydans
3 Biotech, 2018Co-Authors: Ningning Wang, Yuguo ZhengAbstract:Adaptable exploitation of the catalytic potential of membrane-bound d-Sorbitol Dehydrogenase (mSLDH) from Gluconobacter oxydans is desperately needed in the industrial-scale production of miglitol. In the present study, a carbonyl group-dependent colorimetric quantification method was developed for the assay of miglitol key intermediate 6-(N-hydroxyethyl)-amino-6-deoxy-α-l-sorbofuranose (6NSL), and a high-throughput screening process of positive mutants was processed. Combined with several rounds of ultraviolet irradiation mutagenesis and screening procedure, a positive mutant strain G. oxydans ZJB16009 was obtained with significant increase in mSLDH catalytic activity by 1.5-fold, which exhibited an extremely accelerated uptake rate of d-Sorbitol, and the fermentation time was significantly shortened from 22 to 11 h. In a 5-L biotransformation system, 60 g/L substrate N-2-hydroxyethyl glucamine (NHEG) was catalyzed by the resting cells of the mutant strain within 36 h and accumulated 53.6 g/L 6NSL, showing a 33.6% increase in the product yield. Therefore, it was indicated that the established high-throughput screening method could provide a highly efficient platform for the breading of G. oxydans strain for the industrial biosynthesis of miglitol intermediate 6NSL.
Katsuhiko Yoshimoto - One of the best experts on this subject based on the ideXlab platform.
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Redox state-dependent and Sorbitol accumulation-independent diabetic albuminuria in mice with transgene-derived human aldose reductase and Sorbitol Dehydrogenase deficiency
2020Co-Authors: Katsuhiko Yoshimoto, · M Ohta, · E Kudo, · T Yamaoka, · T Tachikawa, · M Moritani, · M Itakura, · K YoshimotoAbstract:Abstract Aims/hypothesis. We investigated the role played by Sorbitol accumulation in the kidney in the development of diabetic albuminuria. Methods. We created mice (hAR-Tg:SDH null) with transgene-derived human aldose reductase and Sorbitol Dehydrogenase (SDH) deficiency, and analysed (i) the contribution of accumulated Sorbitol to urinary albumin excretion rate, and (ii) the effect of the aldose reductase inhibitor, epalrestat, on the diabetic redox state, including decreased renal reduced glutathione concentrations or increased lactate to pyruvate ratios in the diabetic kidney. Results. Compared to littermates, non-diabetic transgenic mice had a 2.6-fold increase in aldose reductase mRNA. In a diabetic group, aldose reductase mRNA in hAR-Tg mice was 2.7-fold higher than in littermates. In the diabetic and non-diabetic groups, hARTg:SDH null mice had the highest Sorbitol content among all four genetic types including hAR-Tg:SDH null, SDH null, hAR-Tg and littermates. The urinary albumin excretion rate in non-diabetic groups was similar in the four genetic types of mouse. In diabetic groups it was greater than in non-diabetic groups, but did not correlate with the Sorbitol content among the four genetic types of mouse. When aldose reductase inhibitor and streptozotocin were given simultaneously at 6 weeks of age, epalrestat prevented diabetic increases in urinary albumin excretion rate and completely prevented diabetic decreases in reduced glutathione concentrations and diabetic increases in lactate to pyruvate ratios, even in the presence of transgenic aldose reductase. Conclusions/interpretation. The degree of diabetic albuminuria in genetically modified mice is dependent on the redox state and independent of polyol accumulation; aldose reductase inhibitor can prevent diabetic albuminuria by normalising diabetic redox changes. [Diabetologi
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redox state dependent and Sorbitol accumulation independent diabetic albuminuria in mice with transgene derived human aldose reductase and Sorbitol Dehydrogenase deficiency
Diabetologia, 2004Co-Authors: M Ohta, Eiji Kudo, Takashi Yamaoka, T Tachikawa, Maki Moritani, Mitsuo Itakura, Katsuhiko YoshimotoAbstract:We investigated the role played by Sorbitol accumulation in the kidney in the development of diabetic albuminuria. We created mice (hAR-Tg:SDH null) with transgene-derived human aldose reductase and Sorbitol Dehydrogenase (SDH) deficiency, and analysed (i) the contribution of accumulated Sorbitol to urinary albumin excretion rate, and (ii) the effect of the aldose reductase inhibitor, epalrestat, on the diabetic redox state, including decreased renal reduced glutathione concentrations or increased lactate to pyruvate ratios in the diabetic kidney. Compared to littermates, non-diabetic transgenic mice had a 2.6-fold increase in aldose reductase mRNA. In a diabetic group, aldose reductase mRNA in hAR-Tg mice was 2.7-fold higher than in littermates. In the diabetic and non-diabetic groups, hAR-Tg:SDH null mice had the highest Sorbitol content among all four genetic types including hAR-Tg:SDH null, SDH null, hAR-Tg and littermates. The urinary albumin excretion rate in non-diabetic groups was similar in the four genetic types of mouse. In diabetic groups it was greater than in non-diabetic groups, but did not correlate with the Sorbitol content among the four genetic types of mouse. When aldose reductase inhibitor and streptozotocin were given simultaneously at 6 weeks of age, epalrestat prevented diabetic increases in urinary albumin excretion rate and completely prevented diabetic decreases in reduced glutathione concentrations and diabetic increases in lactate to pyruvate ratios, even in the presence of transgenic aldose reductase. The degree of diabetic albuminuria in genetically modified mice is dependent on the redox state and independent of polyol accumulation; aldose reductase inhibitor can prevent diabetic albuminuria by normalising diabetic redox changes.
John S Mckinleymckee - One of the best experts on this subject based on the ideXlab platform.
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substrate specificity of sheep liver Sorbitol Dehydrogenase
Biochemical Journal, 1998Co-Authors: Rune I Lindstad, Peter Koll, John S MckinleymckeeAbstract:The substrate specificity of sheep liver Sorbitol Dehydrogenase has been studied by steady-state kinetics over the range pH 7-10. Sorbitol Dehydrogenase stereo-selectively catalyses the reversible NAD-linked oxidation of various polyols and other secondary alcohols into their corresponding ketones. The kinetic constants are given for various novel polyol substrates, including L-glucitol, L-mannitol, L-altritol, D-altritol, D-iditol and eight heptitols, as well as for many aliphatic and aromatic alcohols. The maximum velocities (kcat) and the substrate specificity-constants (kcat/Km) are positively correlated with increasing pH. The enzyme-catalysed reactions occur by a compulsory ordered kinetic mechanism with the coenzyme as the first, or leading, substrate. With many substrates, the rate-limiting step for the overall reaction is the enzyme-NADH product dissociation. However, with several substrates there is a transition to a mechanism with partial rate-limitation at the ternary complex level, especially at low pH. The kinetic data enable the elucidation of new empirical rules for the substrate specificity of Sorbitol Dehydrogenase. The specificity-constants for polyol oxidation vary as a function of substrate configuration with D-xylo> D-ribo > L-xylo > D-lyxo approximately L-arabino > D-arabino > L-lyxo. Catalytic activity with a polyol or an aromatic substrate and various 1-deoxy derivatives thereof varies with -CH2OH > -CH2NH2 > -CH2OCH3 approximately -CH3. The presence of a hydroxyl group at each of the remaining chiral centres of a polyol, apart from the reactive C2, is also nonessential for productive ternary complex formation and catalysis. A predominantly nonpolar enzymic epitope appears to constitute an important structural determinant for the substrate specificity of Sorbitol Dehydrogenase. The existence of two distinct substrate binding regions in the enzyme active site, along with that of the catalytic zinc, is suggested to account for the lack of stereospecificity at C2 in some polyols.
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reversible inhibition of sheep liver Sorbitol Dehydrogenase by the antidiabetogenic drug 2 hydroxymethyl 4 4 n n dimethylaminosulfonyl 1 piperazino pyrimidine
FEBS Letters, 1997Co-Authors: Rune I Lindstad, John S MckinleymckeeAbstract:The mechanism of the inhibition of sheep liver Sorbitol Dehydrogenase by the novel antidiabetogenic drug 2-hydroxymethyl-4-(4-N,N-dimethylaminosulfonyl-1-piperazino)pyrimidine has been investigated by steady-state kinetics over the range pH 5–10. The pyrimidine derivative exhibits mixed inhibition with respect to Sorbitol, fructose and coenzyme, due to the formation of enzyme-inhibitor and enzyme-NAD(H)-inhibitor complexes. The formation of each of the binary and ternary complexes is inhibited by protonation and deprotonation of groups which, in the enzyme-inhibitor complex, have pK values of 6.6 and 8.0, respectively.
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inhibition and activation studies on sheep liver Sorbitol Dehydrogenase
FEBS Journal, 1994Co-Authors: Rune I Lindstad, Leonila F Hermansen, John S MckinleymckeeAbstract:Reversible inhibition and activation, as well as protection against affinity labelling with DL-2-bromo-3-(5-imidazolyl)propionic acid, of sheep liver Sorbitol Dehydrogenase have been studied. The results presented are discussed in terms of enzyme active-site properties and may have potential applications for drug design. Kinetics with mainly Sorbitol competitive inhibitors reveals that aliphatic thiols are generally the most potent inhibitors of enzyme activity. Inhibition and inactivation by heterocyclics parallel that seen previously with Sorbitol Dehydrogenase from other sources as well as with alcohol Dehydrogenase from yeast. However, there are significant differences in relation to the structurally similar horse liver alcohol Dehydrogenase, as the catalytic zinc of Sorbitol Dehydrogenase is more easily removed by chelating molecules. Several aldose reductase inhibitors are shown to also inhibit Sorbitol Dehydrogenase, but at concentrations unlikely to be reached clinically. Enzyme activation has been observed with various compounds, in particular halo-alcohols and detergents. Several inhibitors provide competitive protection against enzyme inactivation by DL-2-bromo-3-(5-imidazolyl)propionic acid. This enables the dissociation constants for binary enzyme-inhibitor complexes to be determined. NADH protects noncompetitively against inactivation. The presence of some binary and ternary enzyme-NADH complexes is indicated from fluorescence emission spectra, as a shift in the fluorescence maximum and intensity is observed due to their formation.
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methylglyoxal and the polyol pathway three carbon compounds are substrates for sheep liver Sorbitol Dehydrogenase
FEBS Letters, 1993Co-Authors: Rune I Lindstad, John S MckinleymckeeAbstract:Methylglyoxal, 1,2-propanediol and glycerol are shown to be substrates for sheep liver Sorbitol Dehydrogenase. With 1,2-propanediol the enzymecatalyzed reaction occurs specifically with the R(−)-enantiomer. The maximum velocities and the specificity constants obtained for the three-carbon substrates are considerably lower than those reported previously for Sorbitol, and suggest that rate-determination is imposed by catalytic steps other than the enzyme-coenzyme product dissociation. The present findings are discussed in terms of substrate specificity and stereospecificity, and may indicate novel aspects of Sorbitol Dehydrogenase function in relation to glucose metabolism and diabetic pathogenesis.
Shohei Yamaki - One of the best experts on this subject based on the ideXlab platform.
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cdna cloning of nad dependent Sorbitol Dehydrogenase from peach fruit and its expression during fruit development
Journal of Horticultural Science & Biotechnology, 2015Co-Authors: Kunio Yamada, N Niwa, Katsuhiro Shiratake, Shohei YamakiAbstract:SummaryFull length cDNA for NAD-dependent Sorbitol Dehydrogenase (NAD-SDH; EC 1.1.1.14) was cloned from peach fruit by reverse transcription polymerase chain reaction (RT-PCR) and 3 9 /5 9 rapid amplification of cDNA ends (RACE).The full-length cDNA consisted of 1457 bp and contained an open reading frame of 1101 bp capable of encoding a protein of 367 amino acids. The deduced amino acid sequence revealed about 77% identity to apple NAD-SDH. Peach NAD-SDH activity based on FW was very strong in immature fruit and declined temporarily, then increased again with fruit maturation. The activity based on the whole fruit showed a peak at 54 and 69.d after flowering (DAF), declined temporarily and increased greatly with fruit maturation. The seasonal change of NAD-dependent Sorbitol Dehydrogenase (NAD-SDH) protein levels based on fresh weight (FW) corresponded to that of the activity based on FW. The typical post translational modification of this enzyme was not indicated. On the other hand, the seasonal change ...
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properties of Sorbitol Dehydrogenase in strawberry fruit and enhancement of the activity by fructose and auxin
Journal of The Japanese Society for Horticultural Science, 2008Co-Authors: Duangsrisai Sutsawat, Kunio Yamada, Katsuhiro Shiratake, Yoshinori Kanayama, Shohei YamakiAbstract:Strawberry, belonging to the Rosaceae family, translocates sucrose as the main carbohydrate to fruit, although many Rosaceae fruit trees use Sorbitol as translocated sugar; however, we have found that strawberry has genes encoding Sorbitol-metabolizing enzymes. In this study, the property of NAD+-dependent Sorbitol Dehydrogenase (NAD-SDH) and the effect of sugars and phytohormones on NAD-SDH activity and its mRNA accumulation were investigated to elucidate Sorbitol metabolism and its regulation. The Km values of NAD-SDH for substrates of fructose and Sorbitol were 78.7 and 7.3 mM, respectively, similar to those of maize, which synthesizes Sorbitol by the reduction of fructose by NAD-SDH. This result suggested that NAD-SDH in strawberry fruit can catalyze the reduction of fructose, but the activity is not enough to accumulate Sorbitol in fruit. Therefore, we investigated the effect of sugars and phytohormones on NAD-SDH activity and the transcript level by adding various sugars and phytohormones to sliced fruit discs. Tissues incubated in 100 mM fructose or Sorbitol stimulated NAD-SDH activity by about 2.5-fold compared with the control. Sucrose also stimulated NAD-SDH activity, but the increase was not as high as Sorbitol or fructose. Of the phytohormones treated, 100 μM indole-3-acetic acid (IAA) had a marked stimulatory effect on NAD-SDH activity, but the other phytohormones (abscisic acid, gibberellic acid and 6-benzyladenine) had no stimulative effect. The mRNA transcript level in all sugar and phytohormone treatments showed no marked increase compared with the control. Thus, the kinetics and induction profiles by phytohormones of NAD-SDH showed that Sorbitol metabolism in strawberry fruit was different from that in apple and Japanese pear fruits.
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presence and expression of nad dependent Sorbitol Dehydrogenase and Sorbitol 6 phosphate Dehydrogenase genes in strawberry
Journal of Horticultural Science & Biotechnology, 2007Co-Authors: S Duangsrisai, Kunio Yamada, Katsuhiro Shiratake, Yoshinori Kanayama, Nancy Bantog, Shohei YamakiAbstract:SummaryThe Sorbitol metabolic pathway in strawberry (Fragaria ananassa Duch. var. ‘Nyoho’) is unclear, despite strawberry belonging to the family Rosaceae. Therefore, the presence or absence of genes for NAD+-dependent Sorbitol Dehydrogenase (NAD-SDH) and Sorbitol-6-phosphate Dehydrogenase (S6PDH) in strawberry, and their levels of expression related to growth were investigated. A full-length cDNA encoding the NAD-SDH gene in strawberry (FaSDH) was obtained using rapid amplification of cDNA ends (RACE). The full-length clone consisted of 1,399 bp and contained a 1,083 bp open reading frame encoding 361 amino acids with a calculated molecular mass of 38.8 kDa, and a predicted isoelectric point (pI) of 6.08. This nucleotide sequence showed 79 – 85% similarity with the NAD-SDH genes of other plants. Its amino acid sequence contained both zinc- and NAD-binding sites, confirming that this sequence belonged to the NAD-SDH gene family. Southern blotting suggested that the genomic copy number of FaSDH was less th...
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molecular evidence of Sorbitol Dehydrogenase in tomato a non rosaceae plant
Phytochemistry, 2005Co-Authors: Kazuhiro Ohta, Shohei Yamaki, Ryo Moriguchi, Koki Kanahama, Yoshinori KanayamaAbstract:The enzyme NAD-dependent Sorbitol Dehydrogenase (SDH) is well characterized in the Rosaceae family of fruit trees, which synthesizes Sorbitol as a translocatable photosynthate. Expressed sequence tags of SDH-like sequences have also been generated from various non-Rosaceae species that do not synthesize Sorbitol as a primary photosynthetic product, but the physiological roles of the encoded proteins in non-Rosaceae plants are unknown. Therefore, we isolated an SDH-like cDNA (SDL) from tomato (Lycopersicon esculentum Mill.). Genomic Southern blot analysis suggested that SDL exists in the tomato genome as a single-copy gene. Northern blot analysis showed that SDL is ubiquitously expressed in tomato plants. Recombinant SDL protein was produced and purified for enzymatic characterization. SDL catalyzed the interconversion of Sorbitol and fructose with NAD (H). SDL showed highest activity for Sorbitol among the several substrates tested. SDL showed no activity with NADP+. Thus, SDL was identified as a SDH, although the Km values and substrate specificity of SDL were significantly different from those of SDH purified from the Japanese pear (Pyrus pyrifolia), a Rosaceae fruit tree. In addition, tomato was transformed with antisense SDL to evaluate the contribution of SDL to SDH activity in tomato. The transformation decreased SDH activity to approximately 50% on average. Taken together, these results provide molecular evidence of SDH in tomato, and SDL was renamed LeSDH.
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purification and characterization of a nad dependent Sorbitol Dehydrogenase from japanese pear fruit
Phytochemistry, 2000Co-Authors: Yasushi Oura, Kunio Yamada, Katsuhiro Shiratake, Shohei YamakiAbstract:Abstract 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 K m 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.
Rune I Lindstad - One of the best experts on this subject based on the ideXlab platform.
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Inhibition of Sorbitol Dehydrogenase by nucleosides and nucleotides
Biochemical and Biophysical Research Communications, 2013Co-Authors: Rune I Lindstad, Knut Teigen, Lars SkjeldalAbstract:Abstract Sorbitol Dehydrogenase inhibitors have been found to prevent, or alleviate, various secondary complications of diabetes mellitus. In the present study, the effects of nucleosides and nucleotides on the rate of Sorbitol oxidation catalyzed by the sheep liver enzyme were studied by steady-state kinetics at pH 7.4. Various such compounds, including ATP and the 2′-deoxy-analogues of ATP, ADP and AMP, reversibly inhibit enzyme activity by formation of enzyme–coenzyme-inhibitor ternary complexes. In each case, no deviations from linearity were seen in the double-reciprocal plots using Sorbitol or NAD+ as the varied substrate and there was a linear relationship between inhibitor concentration and the observed inhibitory effects. Sorbitol was docked into a model of the sheep SDH-NAD+ complex based upon the structure of the human SDH-NAD+ holoenzyme. The resulting structure of the ternary complex of sheep SDH, NAD+ and Sorbitol (PMDB ID code PM 0078068) shows that the reactive C-2 hydroxyl group of Sorbitol is oriented toward the 4′-position of the nicotinamide moiety of the coenzyme, and that the adjacent primary hydroxyl group of Sorbitol interacts with the catalytic zinc. The results indicate that the ribose moiety of the inhibitor structures is an important determinant for the observed effects. Specifically, the 2′-position of the ribose ring exerts an effect with respect to inhibitor potency.
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substrate specificity of sheep liver Sorbitol Dehydrogenase
Biochemical Journal, 1998Co-Authors: Rune I Lindstad, Peter Koll, John S MckinleymckeeAbstract:The substrate specificity of sheep liver Sorbitol Dehydrogenase has been studied by steady-state kinetics over the range pH 7-10. Sorbitol Dehydrogenase stereo-selectively catalyses the reversible NAD-linked oxidation of various polyols and other secondary alcohols into their corresponding ketones. The kinetic constants are given for various novel polyol substrates, including L-glucitol, L-mannitol, L-altritol, D-altritol, D-iditol and eight heptitols, as well as for many aliphatic and aromatic alcohols. The maximum velocities (kcat) and the substrate specificity-constants (kcat/Km) are positively correlated with increasing pH. The enzyme-catalysed reactions occur by a compulsory ordered kinetic mechanism with the coenzyme as the first, or leading, substrate. With many substrates, the rate-limiting step for the overall reaction is the enzyme-NADH product dissociation. However, with several substrates there is a transition to a mechanism with partial rate-limitation at the ternary complex level, especially at low pH. The kinetic data enable the elucidation of new empirical rules for the substrate specificity of Sorbitol Dehydrogenase. The specificity-constants for polyol oxidation vary as a function of substrate configuration with D-xylo> D-ribo > L-xylo > D-lyxo approximately L-arabino > D-arabino > L-lyxo. Catalytic activity with a polyol or an aromatic substrate and various 1-deoxy derivatives thereof varies with -CH2OH > -CH2NH2 > -CH2OCH3 approximately -CH3. The presence of a hydroxyl group at each of the remaining chiral centres of a polyol, apart from the reactive C2, is also nonessential for productive ternary complex formation and catalysis. A predominantly nonpolar enzymic epitope appears to constitute an important structural determinant for the substrate specificity of Sorbitol Dehydrogenase. The existence of two distinct substrate binding regions in the enzyme active site, along with that of the catalytic zinc, is suggested to account for the lack of stereospecificity at C2 in some polyols.
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reversible inhibition of sheep liver Sorbitol Dehydrogenase by the antidiabetogenic drug 2 hydroxymethyl 4 4 n n dimethylaminosulfonyl 1 piperazino pyrimidine
FEBS Letters, 1997Co-Authors: Rune I Lindstad, John S MckinleymckeeAbstract:The mechanism of the inhibition of sheep liver Sorbitol Dehydrogenase by the novel antidiabetogenic drug 2-hydroxymethyl-4-(4-N,N-dimethylaminosulfonyl-1-piperazino)pyrimidine has been investigated by steady-state kinetics over the range pH 5–10. The pyrimidine derivative exhibits mixed inhibition with respect to Sorbitol, fructose and coenzyme, due to the formation of enzyme-inhibitor and enzyme-NAD(H)-inhibitor complexes. The formation of each of the binary and ternary complexes is inhibited by protonation and deprotonation of groups which, in the enzyme-inhibitor complex, have pK values of 6.6 and 8.0, respectively.
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inhibition and activation studies on sheep liver Sorbitol Dehydrogenase
FEBS Journal, 1994Co-Authors: Rune I Lindstad, Leonila F Hermansen, John S MckinleymckeeAbstract:Reversible inhibition and activation, as well as protection against affinity labelling with DL-2-bromo-3-(5-imidazolyl)propionic acid, of sheep liver Sorbitol Dehydrogenase have been studied. The results presented are discussed in terms of enzyme active-site properties and may have potential applications for drug design. Kinetics with mainly Sorbitol competitive inhibitors reveals that aliphatic thiols are generally the most potent inhibitors of enzyme activity. Inhibition and inactivation by heterocyclics parallel that seen previously with Sorbitol Dehydrogenase from other sources as well as with alcohol Dehydrogenase from yeast. However, there are significant differences in relation to the structurally similar horse liver alcohol Dehydrogenase, as the catalytic zinc of Sorbitol Dehydrogenase is more easily removed by chelating molecules. Several aldose reductase inhibitors are shown to also inhibit Sorbitol Dehydrogenase, but at concentrations unlikely to be reached clinically. Enzyme activation has been observed with various compounds, in particular halo-alcohols and detergents. Several inhibitors provide competitive protection against enzyme inactivation by DL-2-bromo-3-(5-imidazolyl)propionic acid. This enables the dissociation constants for binary enzyme-inhibitor complexes to be determined. NADH protects noncompetitively against inactivation. The presence of some binary and ternary enzyme-NADH complexes is indicated from fluorescence emission spectra, as a shift in the fluorescence maximum and intensity is observed due to their formation.
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methylglyoxal and the polyol pathway three carbon compounds are substrates for sheep liver Sorbitol Dehydrogenase
FEBS Letters, 1993Co-Authors: Rune I Lindstad, John S MckinleymckeeAbstract:Methylglyoxal, 1,2-propanediol and glycerol are shown to be substrates for sheep liver Sorbitol Dehydrogenase. With 1,2-propanediol the enzymecatalyzed reaction occurs specifically with the R(−)-enantiomer. The maximum velocities and the specificity constants obtained for the three-carbon substrates are considerably lower than those reported previously for Sorbitol, and suggest that rate-determination is imposed by catalytic steps other than the enzyme-coenzyme product dissociation. The present findings are discussed in terms of substrate specificity and stereospecificity, and may indicate novel aspects of Sorbitol Dehydrogenase function in relation to glucose metabolism and diabetic pathogenesis.