Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: UNIPROT:Q8NEX9 (reductase)
26,410 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Xylose reductase (XR) activity was evaluated in extracts of Candida mogii grown in media containing different concentrations of rice straw hydrolysate. Results of XR activity were compared to xylitol production and a similar behavior was observed for these parameters. Highest values of specific production and productivity were found for xylose reductase (35 U/g of cell and 0.97 U/[g of cell x h], respectively) and for xylitol (5.63 g/g of cell and 0.13 g/[g of cell x h]) in fermentation conducted in medium containing 49.2 g of xylose/L. The maximum value of XR:XD ratio (1.82) was also calculated under this initial xylose concentration with 60 h of fermentation.
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PMID:Activity of xylose reductase from Candida mogii grown in media containing different concentrations of rice straw hydrolysate. 1196 1

Xylose reductase enzyme (EC 1.1.1.21) produced by Candida guilliermondii in sugarcane bagasse was extracted by reversed micelles of N-benzyl-N-dodecyl-N-bis (2-hydroxyethyl) ammonium chloride cationic surfactant. An experimental design was employed to evaluate the influences of the following factors on the enzyme extraction: temperature, cosolvent, and surfactant concentration. A model was used to represent the enzyme recovery and fit of the experimental data. The extraction yielded a total recovery of 130%, and the purity increased 4.8-fold. This study demonstrates that liquid-liquid extraction by reversed micelles is a process able to recover and increase the enzymatic activity and purity of XR produced by C. guilliermondii.
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PMID:Extraction by reversed micelles of the intracellular enzyme xylose reductase. 1196 3

Xylose reductase activity of Candida guilliermondii FTI 20037 was evaluated during xylitol production by fed-batch fermentation of sugarcane bagasse hydrolysate. A 2(4-1) fractional factorial design was used to select process variables. The xylose concentrations in the feeding solution (S(F)) and in the fermentor (S0), the pH, and the aeration rate were selected for optimization of this process, which will be undertaken in the near future. The best experimental result was achieved at S(F) = 45 g/L, S0 = 40 g/L, pH controlled at 6.0, and aeration rate of 1.2 vvm. Under these conditions, the xylose reductase activity was 0.81 U/mg of protein and xylitol production was 26.3 g/L, corresponding to a volumetric productivity of 0.55 g/(L x h) and a xylose xylitol yield factor of 0.68 g/g.
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PMID:Xylose reductase activity of Candida guilliermondii during xylitol production by fed-batch fermentation: selection of process variables. 1201 9

Xylose reductase is a homodimeric oxidoreductase dependent on NADPH or NADH and belongs to the largely monomeric aldo-keto reductase superfamily of proteins. It catalyzes the first step in the assimilation of xylose, an aldose found to be a major constituent monosaccharide of renewable plant hemicellulosic material, into yeast metabolic pathways. It does this by reducing open chain xylose to xylitol, which is reoxidized to xylulose by xylitol dehydrogenase and metabolically integrated via the pentose phosphate pathway. No structure has yet been determined for a xylose reductase, a dimeric aldo-keto reductase or a family 2 aldo-keto reductase. The structures of the Candida tenuis xylose reductase apo- and holoenzyme, which crystallize in spacegroup C2 with different unit cells, have been determined to 2.2 A resolution and an R-factor of 17.9 and 20.8%, respectively. Residues responsible for mediating the novel dimeric interface include Asp-178, Arg-181, Lys-202, Phe-206, Trp-313, and Pro-319. Alignments with other superfamily members indicate that these interactions are conserved in other dimeric xylose reductases but not throughout the remainder of the oligomeric aldo-keto reductases, predicting alternate modes of oligomerization for other families. An arrangement of side chains in a catalytic triad shows that Tyr-52 has a conserved function as a general acid. The loop that folds over the NAD(P)H cosubstrate is disordered in the apo form but becomes ordered upon cosubstrate binding. A slow conformational isomerization of this loop probably accounts for the observed rate-limiting step involving release of cosubstrate. Xylose binding (K(m) = 87 mM) is mediated by interactions with a binding pocket that is more polar than a typical aldo-keto reductase. Modeling of xylose into the active site of the holoenzyme using ordered waters as a guide for sugar hydroxyls suggests a convincing mode of substrate binding.
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PMID:The structure of apo and holo forms of xylose reductase, a dimeric aldo-keto reductase from Candida tenuis. 1210 21

Null mutations in the structural gene encoding phosphoglucose isomerase completely abolish activity of this glycolytic enzyme in Kluyveromyces lactis and Saccharomyces cerevisiae. In S. cerevisiae, the pgi1 null mutation abolishes growth on glucose, whereas K.lactis rag2 null mutants still grow on glucose. It has been proposed that, in the latter case, growth on glucose is made possible by an ability of K. lactis mitochondria to oxidize cytosolic NADPH. This would allow for a re-routing of glucose dissimilation via the pentose-phosphate pathway. Consistent with this hypothesis, mitochondria of S. cerevisiae cannot oxidize NADPH. In the present study, the ability of K. lactis mitochondria to oxidize cytosolic NADPH was experimentally investigated. Respiration-competent mitochondria were isolated from aerobic, glucose-limited chemostat cultures of the wild-type K. lactis strain CBS 2359 and from an isogenic rag2Delta strain. Oxygen-uptake experiments confirmed the presence of a mitochondrial NADPH dehydrogenase in K.lactis. This activity was ca. 2.5-fold higher in the rag2Delta mutant than in the wild-type strain. In contrast to mitochondria from wild-type K. lactis, mitochondria from the rag2Delta mutant exhibited high rates of ethanol-dependent oxygen uptake. Subcellular fractionation studies demonstrated that, in the rag2Delta mutant, a mitochondrial alcohol dehydrogenase was present and that activity of a cytosolic NADPH-dependent 'acetaldehyde reductase' was also increased. These observations indicate that two mechanisms may participate in mitochondrial oxidation of cytosolic NADPH by K. lactis mitochondria: (a) direct oxidation of cytosolic NADPH by a mitochondrial NADPH dehydrogenase; and (b) a two-compartment transhydrogenase cycle involving NADP(+)- and NAD(+)-dependent alcohol dehydrogenases.
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PMID:Two mechanisms for oxidation of cytosolic NADPH by Kluyveromyces lactis mitochondria. 1211 36

Differences between the recombinant xylose-utilizing Saccharomyces cerevisiae strain TMB 3399 and the mutant strain TMB 3400, derived from TMB 3399 and displaying improved ability to utilize xylose, were investigated by using genome-wide expression analysis, physiological characterization, and biochemical assays. Samples for analysis were withdrawn from chemostat cultures. The characteristics of S. cerevisiae TMB 3399 and TMB 3400 grown on glucose and on a mixture of glucose and xylose, as well as of S. cerevisiae TMB 3400 grown on only xylose, were investigated. The strains were cultivated under chemostat conditions at a dilution rate of 0.1 h(-1), with feeds consisting of a defined mineral medium supplemented with 10 g of glucose liter(-1), 10 g of glucose plus 10 g of xylose liter(-1) or, for S. cerevisiae TMB 3400, 20 g of xylose liter(-1). S. cerevisiae TMB 3400 consumed 31% more xylose of a feed containing both glucose and xylose than S. cerevisiae TMB 3399. The biomass yields for S. cerevisiae TMB 3400 were 0.46 g of biomass g of consumed carbohydrate(-1) on glucose and 0.43 g of biomass g of consumed carbohydrate(-1) on xylose. A K(s) value of 33 mM for xylose was obtained for S. cerevisiae TMB 3400. In general, the percentage error was <20% between duplicate microarray experiments originating from independent fermentation experiments. Microarray analysis showed higher expression in S. cerevisiae TMB 3400 than in S. cerevisiae TMB 3399 for (i) HXT5, encoding a hexose transporter; (ii) XKS1, encoding xylulokinase, an enzyme involved in one of the initial steps of xylose utilization; and (iii) SOL3, GND1, TAL1, and TKL1, encoding enzymes in the pentose phosphate pathway. In addition, the transcriptional regulators encoded by YCR020C, YBR083W, and YPR199C were expressed differently in the two strains. Xylose utilization was, however, not affected in strains in which YCR020C was overexpressed or deleted. The higher expression of XKS1 in S. cerevisiae TMB 3400 than in TMB 3399 correlated with higher specific xylulokinase activity in the cell extracts. The specific activity of xylose reductase and xylitol dehydrogenase was also higher for S. cerevisiae TMB 3400 than for TMB 3399, both on glucose and on the mixture of glucose and xylose.
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PMID:Molecular analysis of a Saccharomyces cerevisiae mutant with improved ability to utilize xylose shows enhanced expression of proteins involved in transport, initial xylose metabolism, and the pentose phosphate pathway. 1257 Sep 90

Xylose reductase (XR) from Candida tenuis (CtXR) is a structurally characterized member of family 2 of the aldo-keto reductase (AKR) superfamily of proteins, its family designation being AKR2B5. The enzyme is composed of two identical subunits that contact each other in a largely hydrated, predominantly polar interface. An important question not clearly answered by CtXR structure pertains to the relationship of oligomerization and enzyme activity. In an effort to destabilize the wild-type dimer, the most important secondary structural element of the CtXR interface, the alpha5 helix, was altered by site-directed mutagenesis. Ala-173 and Leu-174 were replaced individually by arginine, and Arg-180 was changed into alanine. A173R and L174R mutants did not fold properly during recombinant protein production in Escherichia coli and could not be isolated. Like the wild type, the R180A mutant is a dimer in solution which does not dissociate into subunits under mild urea conditions (</=2 M). Catalytic efficiency (k(cat)/K(xylose)) and turnover number (k(cat)) of the R180A mutant for NADH-dependent reduction of D-xylose are both approximately 2.5-fold decreased compared to corresponding kinetic parameters of the wild type. Differences in kinetic isotope effects for the mutant (Dk(cat)=1.0; Dk(cat)/K(xylose)=1.9) and the wild type (Dk(cat)=1.5; Dk(cat)/K(xylose)=2.8) suggest subtle changes in catalytic function as result of the mutation. Therefore, altering interactions at the dimer interface may have long range effects that were not predictable from the X-ray structure.
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PMID:Altering dimer contacts in xylose reductase from Candida tenuis by site-directed mutagenesis: structural and functional properties of R180A mutant. 1260 38

Saccharomyces cerevisiae is able to ferment xylose, when engineered with the enzymes xylose reductase (XYL1) and xylitol dehydrogenase (XYL2). However, xylose fermentation is one to two orders of magnitude slower than glucose fermentation. S. cerevisiae has been proposed to have an insufficient capacity of the non-oxidative pentose phosphate pathway (PPP) for rapid xylose fermentation. Strains overproducing the non-oxidative PPP enzymes ribulose 5-phosphate epimerase (EC 5.1.3.1), ribose 5-phosphate ketol isomerase (EC 5.3.1.6), transaldolase (EC 2.2.1.2) and transketolase (EC 2.2.1.1), as well as all four enzymes simultaneously, were compared with respect to xylose and xylulose fermentation with their xylose-fermenting predecessor S. cerevisiae TMB3001, expressing XYL1, XYL2 and only overexpressing XKS1 (xylulokinase). The level of overproduction in S. cerevisiae TMB3026, overproducing all four non-oxidative PPP enzymes, ranged between 4 and 23 times the level in TMB3001. Overproduction of the non-oxidative PPP enzymes did not influence the xylose fermentation rate in either batch cultures of 50 g l(-1) xylose or chemostat cultures of 20 g l(-1) glucose and 20 g l(-1) xylose. The low specific growth rate on xylose was also unaffected. The results suggest that neither of the non-oxidative PPP enzymes has any significant control of the xylose fermentation rate in S. cerevisiae TMB3001. However, the specific growth rate on xylulose increased from 0.02-0.03 for TMB3001 to 0.12 for the strain overproducing only transaldolase (TAL1) and to 0.23 for TMB3026, suggesting that overproducing all four enzymes has a synergistic effect. TMB3026 consumed xylulose about two times faster than TMB30001 in batch culture of 50 g l(-1) xylulose. The results indicate that growth on xylulose and the xylulose fermentation rate are partly controlled by the non-oxidative PPP, whereas control of the xylose fermentation rate is situated upstream of xylulokinase, in xylose transport, in xylose reductase, and/or in the xylitol dehydrogenase.
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PMID:The non-oxidative pentose phosphate pathway controls the fermentation rate of xylulose but not of xylose in Saccharomyces cerevisiae TMB3001. 1270 76

The co-ordinates reported have been submitted to the Protein Data Bank under accession number 1MI3. Xylose reductase (XR; AKR2B5) is an unusual member of aldo-keto reductase superfamily, because it is one of the few able to efficiently utilize both NADPH and NADH as co-substrates in converting xylose into xylitol. In order to better understand the basis for this dual specificity, we have determined the crystal structure of XR from the yeast Candida tenuis in complex with NAD(+) to 1.80 A resolution (where 1 A=0.1 nm) with a crystallographic R -factor of 18.3%. A comparison of the NAD(+)- and the previously determined NADP(+)-bound forms of XR reveals that XR has the ability to change the conformation of two loops. To accommodate both the presence and absence of the 2'-phosphate, the enzyme is able to adopt different conformations for several different side chains on these loops, including Asn(276), which makes alternative hydrogen-bonding interactions with the adenosine ribose. Also critical is the presence of Glu(227) on a short rigid helix, which makes hydrogen bonds to both the 2'- and 3'-hydroxy groups of the adenosine ribose. In addition to changes in hydrogen-bonding of the adenosine, the ribose unmistakably adopts a 3'- endo conformation rather than the 2'- endo conformation seen in the NADP(+)-bound form. These results underscore the importance of tight adenosine binding for efficient use of either NADH or NADPH as a co-substrate in aldo-keto reductases. The dual specificity found in XR is also an important consideration in designing a high-flux xylose metabolic pathway, which may be improved with an enzyme specific for NADH.
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PMID:Structure of xylose reductase bound to NAD+ and the basis for single and dual co-substrate specificity in family 2 aldo-keto reductases. 1273 86

Xylose reductase has been purified to apparent homogeneity from cell extracts of the fungus Cryptococcus flavus grown on D-xylose as carbon source. The enzyme, the first of its kind from the phylum Basidiomycota, is a functional dimer composed of identical subunits of 35.3 kDa mass and requires NADP(H) for activity. Steady-state kinetic parameters for the reaction, D-xylose + NADPH + H(+)<--> xylitol + NADP(+), have been obtained at pH 7.0 and 25 degrees C. The catalytic efficiency for reduction of D-xylose is 150 times that for oxidation of xylitol. This and the 3-fold tighter binding of NADPH than NADP(+) indicate that the enzyme is primed for unidirectional metabolic function in microbial physiology. Kinetic analysis of enzymic reduction of aldehyde substrates differing in hydrophobic and hydrogen bonding capabilities with binary enzyme-NADPH complex has been used to characterize the substrate-binding pocket of xylose reductase. Total transition state stabilization energy derived from bonding with non-reacting sugar hydroxyls is approximately 15 kJ/mol, with a major contribution of 5-8 kJ/mol made by interactions with the C-2(R) hydroxy group. The aldehyde binding site is approximately 1.2 times more hydrophobic than n-octanol and can accommodate linear alkyl chains of <or=6 carbons. Hydrophobic interactions provide a total binding energy of approximately 10 kJ/mol. Specificity for the aldehyde substrate is achieved through large decreases in apparent K(m) ( approximately 100-fold) and smaller but significant increases in turnover number ( approximately 5-fold). We observed up to 250-fold preference of xylose reductase for reaction with pyridine carbaldehydes, 4-nitro-benzaldehyde, and alpha-oxo-aldehydes over reaction with D-xylose, perhaps reflecting a secondary role of this enzyme in detoxication metabolism of reactive endogenous aldehydes and compounds of xenobiotic origin.
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PMID:Xylose reductase from the Basidiomycete fungus Cryptococcus flavus: purification, steady-state kinetic characterization, and detailed analysis of the substrate binding pocket using structure-activity relationships. 1276 4


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