D-Xylulose Reductase: An enzyme that plays a role in the PENTOSES and GLUCURONATES interconversion pathway by catalyzing the oxidation of XYLITOL to D-xylulose. This enzyme has been found to be specific for NAD+.Xylulose: A 5-carbon keto sugar.Xylitol: A five-carbon sugar alcohol derived from XYLOSE by reduction of the carbonyl group. It is as sweet as sucrose and used as a noncariogenic sweetener.Pentoses: A class of carbohydrates that contains five carbon atoms.Fructokinases: A class of enzymes that catalyzes the phosphorylation of fructose in the presence of ATP. EC 2.7.1.-.PentosephosphatesXyloseSugar Alcohols: Polyhydric alcohols having no more than one hydroxy group attached to each carbon atom. They are formed by the reduction of the carbonyl group of a sugar to a hydroxyl group.(From Dorland, 28th ed)Sugar Alcohol Dehydrogenases: Reversibly catalyzes the oxidation of a hydroxyl group of sugar alcohols to form a keto sugar, aldehyde or lactone. Any acceptor except molecular oxygen is permitted. Includes EC 1.1.1.; EC 1.1.2. and EC 1.1.99.Gluconobacter oxydans: A rod-shaped to ellipsoidal, gram-negative bacterium which oxidizes ethanol to acetic acid and prefers sugar-enriched environments. (From Bergey's Manual of Determinative Bacteriology, 9th ed)Carbohydrate Epimerases: Enzymes that catalyze the epimerization of chiral centers within carbohydrates or their derivatives. EC 5.1.3.ArabinoseAldose-Ketose Isomerases: Enzymes that catalyze the interconversion of aldose and ketose compounds.Nitrate Reductases: Oxidoreductases that are specific for the reduction of NITRATES.Hydroxymethylglutaryl CoA Reductases: Enzymes that catalyze the reversible reduction of alpha-carboxyl group of 3-hydroxy-3-methylglutaryl-coenzyme A to yield MEVALONIC ACID.Ribonucleotide ReductasesCytochrome-B(5) Reductase: A FLAVOPROTEIN oxidoreductase that occurs both as a soluble enzyme and a membrane-bound enzyme due to ALTERNATIVE SPLICING of a single mRNA. The soluble form is present mainly in ERYTHROCYTES and is involved in the reduction of METHEMOGLOBIN. The membrane-bound form of the enzyme is found primarily in the ENDOPLASMIC RETICULUM and outer mitochondrial membrane, where it participates in the desaturation of FATTY ACIDS; CHOLESTEROL biosynthesis and drug metabolism. A deficiency in the enzyme can result in METHEMOGLOBINEMIA.Nitrite Reductases: A group of enzymes that oxidize diverse nitrogenous substances to yield nitrite. (Enzyme Nomenclature, 1992) EC 1.Glutathione Reductase: Catalyzes the oxidation of GLUTATHIONE to GLUTATHIONE DISULFIDE in the presence of NADP+. Deficiency in the enzyme is associated with HEMOLYTIC ANEMIA. Formerly listed as EC 18.104.22.168.FMN Reductase: An enzyme that utilizes NADH or NADPH to reduce FLAVINS. It is involved in a number of biological processes that require reduced flavin for their functions such as bacterial bioluminescence. Formerly listed as EC 22.214.171.124 and EC 126.96.36.199.Thioredoxin-Disulfide Reductase: A FLAVOPROTEIN enzyme that catalyzes the oxidation of THIOREDOXINS to thioredoxin disulfide in the presence of NADP+. It was formerly listed as EC 188.8.131.52Phosphotransferases: A rather large group of enzymes comprising not only those transferring phosphate but also diphosphate, nucleotidyl residues, and others. These have also been subdivided according to the acceptor group. (From Enzyme Nomenclature, 1992) EC 2.7.NADPH-Ferrihemoprotein Reductase: A flavoprotein that catalyzes the reduction of heme-thiolate-dependent monooxygenases and is part of the microsomal hydroxylating system. EC 184.108.40.206.Ferredoxin-NADP Reductase: An enzyme that catalyzes the oxidation and reduction of FERREDOXIN or ADRENODOXIN in the presence of NADP. EC 220.127.116.11 was formerly listed as EC 18.104.22.168 and EC 22.214.171.124.Oxidoreductases: The class of all enzymes catalyzing oxidoreduction reactions. The substrate that is oxidized is regarded as a hydrogen donor. The systematic name is based on donor:acceptor oxidoreductase. The recommended name will be dehydrogenase, wherever this is possible; as an alternative, reductase can be used. Oxidase is only used in cases where O2 is the acceptor. (Enzyme Nomenclature, 1992, p9)Cytochrome ReductasesTetrahydrofolate Dehydrogenase: An enzyme of the oxidoreductase class that catalyzes the reaction 7,8-dihyrofolate and NADPH to yield 5,6,7,8-tetrahydrofolate and NADPH+, producing reduced folate for amino acid metabolism, purine ring synthesis, and the formation of deoxythymidine monophosphate. Methotrexate and other folic acid antagonists used as chemotherapeutic drugs act by inhibiting this enzyme. (Dorland, 27th ed) EC 126.96.36.199.Methylenetetrahydrofolate Reductase (NADPH2): A flavoprotein amine oxidoreductase that catalyzes the reversible conversion of 5-methyltetrahydrofolate to 5,10-methylenetetrahydrofolate. This enzyme was formerly classified as EC 188.8.131.52.Nitrate Reductase (NADH): An NAD-dependent enzyme that catalyzes the oxidation of nitrite to nitrate. It is a FLAVOPROTEIN that contains IRON and MOLYBDENUM and is involved in the first step of nitrate assimilation in PLANTS; FUNGI; and BACTERIA. It was formerly classified as EC 184.108.40.206.Methionine Sulfoxide Reductases: Reductases that catalyze the reaction of peptide-L-methionine -S-oxide + thioredoxin to produce peptide-L-methionine + thioredoxin disulfide + H(2)O.NADP: Nicotinamide adenine dinucleotide phosphate. A coenzyme composed of ribosylnicotinamide 5'-phosphate (NMN) coupled by pyrophosphate linkage to the 5'-phosphate adenosine 2',5'-bisphosphate. It serves as an electron carrier in a number of reactions, being alternately oxidized (NADP+) and reduced (NADPH). (Dorland, 27th ed)
PentosuriaXylitolApioseFructokinase: Fructokinase (/fruc•to•ki•nase/ [-ki´nas]) (), also known as D-fructokinase or D-fructose (D-mannose) kinase,DBGET ENZYME: 2.7.D-xylose reductase: D-xylose reductase (, XylR, XyrA, msXR, dsXR, monospecific xylose reductase, dual specific xylose reductase, NAD(P)H-dependent xylose reductase, xylose reductase) is an enzyme with system name xylitol:NAD(P)+ oxidoreductase. This enzyme catalyses the following chemical reactionD-arabitol-phosphate dehydrogenase: D-arabitol-phosphate dehydrogenase (, APDH, D-arabitol 1-phosphate dehydrogenase, D-arabitol 5-phosphate dehydrogenase) is an enzyme with system name D-arabitol-phosphate:NAD+ oxidoreductase. This enzyme catalyses the following chemical reactionL-arabinose operon: The -arabinose operon, also called the ara or araBAD operon, is an operon that encodes enzymes needed for the catabolism of arabinose in Escherichia coli. It has both positive and negative regulation and is activated allosterically.Davidaster rubiginosusNitrate reductase (NADPH): Nitrate reductase (NADPH) (, assimilatory nitrate reductase, assimilatory reduced nicotinamide adenine dinucleotide phosphate-nitrate reductase, NADPH-nitrate reductase, assimilatory NADPH-nitrate reductase, triphosphopyridine nucleotide-nitrate reductase, NADPH:nitrate reductase, nitrate reductase (NADPH2), NADPH2:nitrate oxidoreductase) is an enzyme with system name nitrite:NADP+ oxidoreductase. This enzyme catalises the following chemical reactionHMG-CoA reductase: HMG-CoA reductase (3-hydroxy-3-methyl-glutaryl-CoA reductase, officially abbreviated HMGCR) is the rate-controlling enzyme (NADH-dependent, ; NADPH-dependent, ) of the mevalonate pathway, the metabolic pathway that produces cholesterol and other isoprenoids. Normally in mammalian cells this enzyme is suppressed by cholesterol derived from the internalization and degradation of low density lipoprotein (LDL) via the LDL receptor as well as oxidized species of cholesterol.Ribonucleotide: In biochemistry, a ribonucleotide or ribotide is a nucleotide containing ribose as its pentose component. It is considered a molecular precursor of nucleic acids.Cytochrome b5 reductaseFMN reductase (NADPH): FMN reductase (NADPH) (, FRP, flavin reductase P, SsuE) is an enzyme with system name FMNH2:NADP+ oxidoreductase. This enzyme catalyses the following chemical reaction:Thioredoxin reductasePhosphotransferase: Phosphotransferases are a category of enzymes (EC number 2.7) that catalyze phosphorylation reactions.FnrS RNA: FnrS RNA is a family of Hfq-binding small RNA whose expression is upregulated in response to anaerobic conditions. It is named FnrS because its expression is strongly dependent on fumarate and nitrate reductase regulator (FNR), a direct oxygen availability sensor.Glucose-methanol-choline oxidoreductase family: In molecular biology, the glucose-methanol-choline oxidoreductase family (GMC oxidoreductase) is a family of enzymes with oxidoreductase activity.Flavoprotein pyridine nucleotide cytochrome reductases: A:229-424 A:229-424 A:985-1214Dihydrofolate reductaseMethylenetetrahydrofolate reductase: Methylene tetrahydrofolate reductase (MTHFR) is the rate-limiting enzyme in the methyl cycle, and it is encoded by the MTHFR gene. Methylenetetrahydrofolate reductase catalyzes the conversion of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate, a cosubstrate for homocysteine remethylation to methionine.Methionine sulfoxide
(1/40) Evidence that the gene YLR070c of Saccharomyces cerevisiae encodes a xylitol dehydrogenase.
The open reading frame YLR070c of Saccharomyces cerevisiae has high sequence similarity to S. cerevisiae sorbitol dehydrogenase and to xylitol dehydrogenase of Pichia stipitis. Overexpression of this open reading frame in S. cerevisiae resulted in xylitol dehydrogenase activity. The enzyme is specific for NADH. The following Michaelis constants were estimated: D-xylulose, 1.1 mM; NADH, 240 microM (at pH 7.0); xylitol, 25 mM; NAD, 100 microM (at pH 9.0). Xylitol dehydrogenase activity with the same kinetic properties can also be induced by xylose in wild type S. cerevisiae cells. (+info)
(2/40) Xylulokinase overexpression in two strains of Saccharomyces cerevisiae also expressing xylose reductase and xylitol dehydrogenase and its effect on fermentation of xylose and lignocellulosic hydrolysate.
Fermentation of the pentose sugar xylose to ethanol in lignocellulosic biomass would make bioethanol production economically more competitive. Saccharomyces cerevisiae, an efficient ethanol producer, can utilize xylose only when expressing the heterologous genes XYL1 (xylose reductase) and XYL2 (xylitol dehydrogenase). Xylose reductase and xylitol dehydrogenase convert xylose to its isomer xylulose. The gene XKS1 encodes the xylulose-phosphorylating enzyme xylulokinase. In this study, we determined the effect of XKS1 overexpression on two different S. cerevisiae host strains, H158 and CEN.PK, also expressing XYL1 and XYL2. H158 has been previously used as a host strain for the construction of recombinant xylose-utilizing S. cerevisiae strains. CEN.PK is a new strain specifically developed to serve as a host strain for the development of metabolic engineering strategies. Fermentation was carried out in defined and complex media containing a hexose and pentose sugar mixture or a birch wood lignocellulosic hydrolysate. XKS1 overexpression increased the ethanol yield by a factor of 2 and reduced the xylitol yield by 70 to 100% and the final acetate concentrations by 50 to 100%. However, XKS1 overexpression reduced the total xylose consumption by half for CEN.PK and to as little as one-fifth for H158. Yeast extract and peptone partly restored sugar consumption in hydrolysate medium. CEN.PK consumed more xylose but produced more xylitol than H158 and thus gave lower ethanol yields on consumed xylose. The results demonstrate that strain background and modulation of XKS1 expression are important for generating an efficient xylose-fermenting recombinant strain of S. cerevisiae. (+info)
(3/40) Novel enzymatic method for the production of xylitol from D-arabitol by Gluconobacter oxydans.
Microorganisms capable of producing xylitol from D-arabitol were screened for. Of the 420 strains tested, three bacteria, belonging to the genera Acetobacter and Gluconobacter, produced xylitol from D-arabitol when intact cells were used as the enzyme source. Among them, Gluconobacter oxydans ATCC 621 produced 29.2 g/l xylitol from 52.4 g/l D-arabitol after incubation for 27 h. The production of xylitol was increased by the addition of 5% (v/v) ethanol and 5 g/l D-glucose to the reaction mixture. Under these conditions, 51.4 g/l xylitol was obtained from 52.4 g/l D-arabitol, a yield of 98%, after incubation for 27 h. This conversion consisted of two successive reactions, conversion of D-arabitol to D-xylulose by a membrane-bound D-arabitol dehydrogenase, and conversion of D-xylulose to xylitol by a soluble NAD-dependent xylitol dehydrogenase. Use of disruptants of the membrane-bound alcohol dehydrogenase genes suggested that NADH was generated via NAD-dependent soluble alcohol dehydrogenase. (+info)
(4/40) Evolutionary engineering of Saccharomyces cerevisiae for anaerobic growth on xylose.
Xylose utilization is of commercial interest for efficient conversion of abundant plant material to ethanol. Perhaps the most important ethanol-producing organism, Saccharomyces cerevisiae, however, is incapable of xylose utilization. While S. cerevisiae strains have been metabolically engineered to utilize xylose, none of the recombinant strains or any other naturally occurring yeast has been able to grow anaerobically on xylose. Starting with the recombinant S. cerevisiae strain TMB3001 that overexpresses the xylose utilization pathway from Pichia stipitis, in this study we developed a selection procedure for the evolution of strains that are capable of anaerobic growth on xylose alone. Selection was successful only when organisms were first selected for efficient aerobic growth on xylose alone and then slowly adapted to microaerobic conditions and finally anaerobic conditions, which indicated that multiple mutations were necessary. After a total of 460 generations or 266 days of selection, the culture reproduced stably under anaerobic conditions on xylose and consisted primarily of two subpopulations with distinct phenotypes. Clones in the larger subpopulation grew anaerobically on xylose and utilized both xylose and glucose simultaneously in batch culture, but they exhibited impaired growth on glucose. Surprisingly, clones in the smaller subpopulation were incapable of anaerobic growth on xylose. However, as a consequence of their improved xylose catabolism, these clones produced up to 19% more ethanol than the parental TMB3001 strain produced under process-like conditions from a mixture of glucose and xylose. (+info)
(5/40) Cloning of the xylitol dehydrogenase gene from Gluconobacter oxydans and improved production of xylitol from D-arabitol.
Xylitol dehydrogenase (XDH) was purified from the cytoplasmic fraction of Gluconobacter oxydans ATCC 621. The purified enzyme reduced D-xylulose to xylitol in the presence of NADH with an optimum pH of around 5.0. Based on the determined NH2-terminal amino acid sequence, the gene encoding xdh was cloned, and its identity was confirmed by expression in Escherichia coli. The xdh gene encodes a polypeptide composed of 262 amino acid residues, with an estimated molecular mass of 27.8 kDa. The deduced amino acid sequence suggested that the enzyme belongs to the short-chain dehydrogenase/reductase family. Expression plasmids for the xdh gene were constructed and used to produce recombinant strains of G. oxydans that had up to 11-fold greater XDH activity than the wild-type strain. When used in the production of xylitol from D-arabitol under controlled aeration and pH conditions, the strain harboring the xdh expression plasmids produced 57 g/l xylitol from 225 g/l D-arabitol, whereas the control strain produced 27 g/l xylitol. These results demonstrated that increasing XDH activity in G. oxydans improved xylitol productivity. (+info)
(6/40) D-xylose metabolism in Hypocrea jecorina: loss of the xylitol dehydrogenase step can be partially compensated for by lad1-encoded L-arabinitol-4-dehydrogenase.
With the goal of the genetic characterization of the D-xylose pathway in Hypocrea jecorina (anamorph: Trichoderma reesei), we cloned the xdh1 gene, encoding NAD-xylitol dehydrogenase, which catalyzes the second step of fungal D-xylose catabolism. This gene encodes a 363-amino-acid protein which has a mass of 38 kDa, belongs to the zinc-containing alcohol dehydrogenase family, exhibits high sequence identity to the published sequences of xylitol dehydrogenases from yeast origins, but contains a second, additional binding site for Zn2+. The enzyme catalyzed the NAD-dependent oxidation of xylitol and D-sorbitol and the NADH-dependent reduction of D-xylulose and D-fructose. No activity was observed with NADP, L-arabinose, or L-arabinitol. A single 1.4-kb transcript was formed during growth on xylan, D-xylose, L-arabinose, L-arabinitol and, at a lower abundance, xylitol, D-galactose, galactitol, and lactose but not on D-glucose and glycerol. xdh1 deletion mutants exhibited 50% reduced growth rates on D-xylose, whereas growth rates on xylitol remained unaltered. These mutants contained 30% of the xylitol dehydrogenase activity of the parent strain, indicating the presence of a second xylitol dehydrogenase. This activity was shown to be due to lad1-encoded L-arabinitol-4-dehydrogenase, because H. jecorina xdh1 lad1 double-deletion strains failed to grow on D-xylose or xylitol. In contrast, lad1 deletion strains of H. jecorina grew normally on these carbon sources. These results show that H. jecorina contains a single xylitol dehydrogenase which is encoded by xdh1 and is involved in the metabolism of D-xylose and that lad1-encoded L-arabinitol-4-dehydrogenase can compensate for it partially in mutants with a loss of xdh1 function. (+info)
(7/40) Endogenous xylose pathway in Saccharomyces cerevisiae.
The baker's yeast Saccharomyces cerevisiae is generally classified as a non-xylose-utilizing organism. We found that S. cerevisiae can grow on D-xylose when only the endogenous genes GRE3 (YHR104w), coding for a nonspecific aldose reductase, and XYL2 (YLR070c, ScXYL2), coding for a xylitol dehydrogenase (XDH), are overexpressed under endogenous promoters. In nontransformed S. cerevisiae strains, XDH activity was significantly higher in the presence of xylose, but xylose reductase (XR) activity was not affected by the choice of carbon source. The expression of SOR1, encoding a sorbitol dehydrogenase, was elevated in the presence of xylose as were the genes encoding transketolase and transaldolase. An S. cerevisiae strain carrying the XR and XDH enzymes from the xylose-utilizing yeast Pichia stipitis grew more quickly and accumulated less xylitol than did the strain overexpressing the endogenous enzymes. Overexpression of the GRE3 and ScXYL2 genes in the S. cerevisiae CEN.PK2 strain resulted in a growth rate of 0.01 g of cell dry mass liter(-1) h(-1) and a xylitol yield of 55% when xylose was the main carbon source. (+info)
(8/40) Complete reversal of coenzyme specificity of xylitol dehydrogenase and increase of thermostability by the introduction of structural zinc.
Pichia stipitis NAD(+)-dependent xylitol dehydrogenase (XDH), a medium-chain dehydrogenase/reductase, is one of the key enzymes in ethanol fermentation from xylose. For the construction of an efficient biomass-ethanol conversion system, we focused on the two areas of XDH, 1) change of coenzyme specificity from NAD(+) to NADP(+) and 2) thermostabilization by introducing an additional zinc atom. Site-directed mutagenesis was used to examine the roles of Asp(207), Ile(208), Phe(209), and Asn(211) in the discrimination between NAD(+) and NADP(+). Single mutants (D207A, I208R, F209S, and N211R) improved 5 approximately 48-fold in catalytic efficiency (k(cat)/K(m)) with NADP(+) compared with the wild type but retained substantial activity with NAD(+). The double mutants (D207A/I208R and D207A/F209S) improved by 3 orders of magnitude in k(cat)/K(m) with NADP(+), but they still preferred NAD(+) to NADP(+). The triple mutant (D207A/I208R/F209S) and quadruple mutant (D207A/I208R/F209S/N211R) showed more than 4500-fold higher values in k(cat)/K(m) with NADP(+) than the wild-type enzyme, reaching values comparable with k(cat)/K(m) with NAD(+) of the wild-type enzyme. Because most NADP(+)-dependent XDH mutants constructed in this study decreased the thermostability compared with the wild-type enzyme, we attempted to improve the thermostability of XDH mutants by the introduction of an additional zinc atom. The introduction of three cysteine residues in wild-type XDH gave an additional zinc-binding site and improved the thermostability. The introduction of this mutation in D207A/I208R/F209S and D207A/I208R/F209S/N211R mutants increased the thermostability and further increased the catalytic activity with NADP(+). (+info)