Hyperproduction of tryptophan by Corynebacterium glutamicum with the modified pentose phosphate pathway.
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A classically derived tryptophan-producing Corynebacterium glutamicum strain was recently significantly improved both by plasmid-mediated amplification of the genes for the rate-limiting enzymes in the terminal pathways and by construction of a plasmid stabilization system so that it produced more tryptophan. This engineered strain, KY9218 carrying pKW9901, produced 50 g of tryptophan per liter from sucrose after 80 h in fed-batch cultivation without antibiotic pressure. Analysis of carbon balances showed that at the late stage of the fermentation, tryptophan yield decreased with a concomitant increase in CO2 yield, suggesting a transition in the distribution of carbon flow from aromatic biosynthesis toward the tricarboxylic acid cycle via glycolysis. To circumvent this transition by increasing the supply of erythrose 4-phosphate, a direct precursor of aromatic biosynthesis, the transketolase gene of C. glutamicum was coamplified in the engineered strain by using low- and high-copy-number plasmids which were compatible with the resident plasmid pKW9901. The presence of the gene in low copy numbers contributed to improvement of tryptophan yield, especially at the late stage, and led to accumulation of more tryptophan (57 g/liter) than did its absence, while high-copy-number amplification of the gene resulted in a tryptophan production level even lower than that resulting from the absence of the gene due to reduced growth and sugar consumption. In order to assemble all the cloned genes onto a low-copy-number plasmid, the high-copy-number origin of pKW9901 was replaced with the low-copy-number one, generating low-copy-number plasmid pSW9911, and the transketolase gene was inserted to yield pIK9960. The pSW9911-carrying producer showed almost the same fermentation profiles as the pKW9901 carrier in fed-batch cultivation without antibiotic pressure. Under the same culture conditions, however, the pIK9960 carrier achieved a final tryptophan titer of 58 g/liter, which represented a 15% enhancement over the titers achieved by the pKW9901 and pSW9911 carriers. (+info)
The non-oxidative degradation of ascorbic acid at physiological conditions.
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The degradation of L-ascorbate (AsA) and its primary oxidation products, L-dehydroascorbate (DHA) and 2,3-L-diketogulonate (2, 3-DKG) were studied under physiological conditions. Analysis determined that L-erythrulose (ERU) and oxalate were the primary degradation products of ASA regardless of which compound was used as the starting material. The identification of ERU was determined by proton decoupled (13)C-nuclear magnetic resonance spectroscopy, and was quantified by high performance liquid chromatography, and enzymatic analysis. The molar yield of ERU from 2,3-DKG at pH 7.0 37 degrees C and limiting O(2)97%. This novel ketose product of AsA degradation, was additionally qualitatively identified by gas-liquid chromatography, and by thin layer chromatography. ERU is an extremely reactive ketose, which rapidly glycates and crosslinks proteins, and therefore may mediate the AsA-dependent modification of protein (ascorbylation) seen in vitro, and also proposed to occur in vivo in human lens during diabetic and age-onset cataract formation. (+info)
Site-directed mutation of noncatalytic residues of Thermobifida fusca exocellulase Cel6B.
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Fifteen mutant genes in six loop residues and eight mutant genes in five conserved noncatalytic active site residues of Thermobifida fusca Cel6B were constructed, cloned and expressed in Escherichia coli or Streptomyces lividans. The mutant enzymes were assayed for catalytic activity on carboxymethyl cellulose (CMC), swollen cellulose (SC), filter paper (FP), and bacterial microcrystalline cellulose (BMCC) as well as cellotetraose, cellopentaose, and 2, 4-dinitrophenyl-beta-D-cellobioside. They were also assayed for ligand binding, enzyme processivity, thermostability, and cellobiose feedback inhibition. Two double Cys mutations that formed disulfide bonds across two tunnel forming loops were found to significantly weaken binding to ligands, lower all activities, and processivity, demonstrating that the movement of these loops is important but not essential for Cel6B function. Two single mutant enzymes, G234S and G284P, had higher activity on SC and FP, and the double mutant enzyme had threefold and twofold higher activity on these substrates, respectively. However, synergism with endocellulase T. fusca Cel5A was not increased with these mutant enzymes. All mutant enzymes with lower activity on filter paper, BMCC, and SC had lower processivity. This trend was not true for CMC, suggesting that processivity in Cel6B is a key factor in the hydrolysis of insoluble and crystalline cellulose. Three mutations (E495D, H326A and W329C) located near putative glycosyl substrate subsites -2, +1 and +2, were found to significantly increase resistance to cellobiose feedback inhibition. Both the A229V and L230C mutations specifically decreased activity on BMCC, suggesting that BMCC hydrolysis has a different rate limiting step than the other substrates. Most of the mutant enzymes had reduced thermostability although Cel6B G234S maintained wild-type thermostability. The properties of the different mutant enzymes provide insight into the catalytic mechanism of Cel6B. (+info)
Chemical etiology of nucleic acid structure: the alpha-threofuranosyl-(3'-->2') oligonucleotide system.
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TNAs [(L)-alpha-threofuranosyl oligonucleotides] containing vicinally connected (3'-->2') phosphodiester bridges undergo informational base pairing in antiparallel strand orientation and are capable of cross-pairing with RNA and DNA. Being derived from a sugar containing only four carbons, TNA is structurally the simplest of all potentially natural oligonucleotide-type nucleic acid alternatives studied thus far. This, along with the base-pairing properties of TNA, warrants close scrutiny of the system in the context of the problem of RNA's origin. (+info)
Inhibitors of advanced glycation end product-associated protein cross-linking.
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The reaction of lens proteins with sugars over time results in the formation of protein-bound advanced glycation end products (AGEs). The most damaging element of AGE formation may be the synthesis of protein-protein cross-links in long-lived proteins, such as collagen or lens crystallins. A quantitative cross-linking assay, involving the sugar-dependent incorporation of [U-(14)C]lysine into protein, was employed to determine the efficacy of a variety of potential cross-linking inhibitors. Reaction mixtures contained 5.0 mM L-threose, 2.5 microCi [(14)C]lysine (1.0 mCi/mmole), 5.0 mg/ml bovine lens proteins, 0-10 mM inhibitor and 1.0 mM DTPA in 100 mM phosphate buffer, pH 7.0. Of 17 potential inhibitors tested, 11 showed 50% inhibition or less at 10 mM. The dicarbonyl-reactive compounds 2-aminoguanidine, semicarbazide and o-phenylenediamine inhibited 50% at 2.0 mM, whereas 10 mM dimethylguanidine had no effect. Several amino acids failed to compete effectively with [(14)C]lysine in the cross-linking assay; however, cysteine inhibited 50% at 1.0 mM. This was likely due to the sulfhydryl group of cysteine, because 3-mercaptopropionic acid and reduced glutathione exhibited similar activity. Sodium metabisulfite had the highest activity, inhibiting 50% at only 0.1-0.2 mM. Protein dimer formation, as determined by SDS-PAGE, was inhibited in a quantitatively similar manner. The dicarbonyl-reactive inhibitors and the sulfur-containing compounds produced similar inhibition curves for [(14)C]lysine incorporation over a 3 week assay with 250 mM glucose. A much lesser effect was observed on either the incorporation of [(14)C]glucose, or on fluorophore formation (360/420 nm), suggesting that non-cross-link fluorophores were also formed. The inhibitor data were consistent with cross-linking by a dicarbonyl intermediate. This was supported by the fact that the inhibitors were uniformly less effective when the 5.0 mM threose was replaced by either 3.0 mM 3-deoxythreosone or 3.0 mM threosone. (+info)
Crosslinking by advanced glycation end products increases the stiffness of the collagen network in human articular cartilage: a possible mechanism through which age is a risk factor for osteoarthritis.
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OBJECTIVE: Age is an important risk factor for osteoarthritis (OA). During aging, nonenzymatic glycation results in the accumulation of advanced glycation end products (AGEs) in cartilage collagen. We studied the effect of AGE crosslinking on the stiffness of the collagen network in human articular cartilage. METHODS: To increase AGE levels, human adult articular cartilage was incubated with threose. The stiffness of the collagen network was measured as the instantaneous deformation (ID) of the cartilage and as the change in tensile stress in the collagen network as a function of hydration (osmotic stress technique). AGE levels in the collagen network were determined as: Nepsilon-(carboxy[m]ethyl)lysine, pentosidine, amino acid modification (loss of arginine and [hydroxy-]lysine), AGE fluorescence (360/460 nm), and digestibility by bacterial collagenase. RESULTS: Incubation of cartilage with threose resulted in a dose-dependent increase in AGEs and a concomitant decrease in ID (r = -0.81, P < 0.001; up to a 40% decrease at 200 mM threose), i.e., increased stiffness, which was confirmed by results from the osmotic stress technique. The decreased ID strongly correlated with AGE levels (e.g., AGE fluorescence r = -0.81, P < 0.0001). Coincubation with arginine or lysine (glycation inhibitors) attenuated the threose-induced decrease in ID (P < 0.05). CONCLUSION: Increasing cartilage AGE crosslinking by in vitro incubation with threose resulted in increased stiffness of the collagen network. Increased stiffness by AGE crosslinking may contribute to the age-related failure of the collagen network in human articular cartilage to resist damage. Thus, the age-related accumulation of AGE crosslinks presents a putative molecular mechanism whereby age is a predisposing factor for the development of OA. (+info)
L-erythrulose production by oxidative fermentation is catalyzed by PQQ-containing membrane-bound dehydrogenase.
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Thermotolerant Gluconobacter frateurii CHM 43 was selected for L-erythrulose production from mesoerythritol at higher temperatures. Growing cells and the membrane fraction of the strain rapidly oxidized mesoerythritol to L-erythrulose irreversibly with almost 100% of recovery at 37 degrees C. L-Erythrulose was also produced efficiently by the resting cells at 37 degrees C with 85% recovery. The enzyme responsible for mesoerythritol oxidation was found to be located in the cytoplasmic membrane of the organism. The EDTA-resolved enzyme required PQQ and Ca2+ for L-erythrulose formation, suggesting that the enzyme catalyzing meso-erythritol oxidation was a quinoprotein. Quinoprotein membrane-bound mesoerythritol dehydrogenase (QMEDH) was solubilized and purified to homogeneity. The purified enzyme showed a single band in SDS-PAGE of which the molecular mass corresponded to 80 kDa. The optimum pH of QMEDH was found at pH 5.0. The Michaelis constant of the enzyme was found to be 25 mM for meso-erythritol as the substrate. QMEDH showed a broad substrate specificity toward C3-C6 sugar alcohols in which the erythro form of two hydroxy groups existed adjacent to a primary alcohol group. On the other hand, the cytosolic NAD-denpendent meso-erythritol dehydrogenase (CMEDH) of the same organism was purified to a crystalline state. CMEDH showed a molecular mass of 60 kDa composed of two identical subunits, and an apparent sedimentation constant was 3.6 s. CMEDH catalyzed oxidoreduction between mesoerythritol and L-erythrulose. The oxidation reaction was observed to be reversible in the presence of NAD at alkaline pHs such as 9.0-10.5. L-Erythrulose reduction was found at pH 6.0 with NADH as coenzyme. Judging from the catalytic properties, the NAD-dependent enzyme in the cytosolic fraction was regarded as a typical pentitol dehydrogenase of NAD-dependent and the enzyme was independent of the oxidative fermentation of L-erythrulose production. (+info)
Anacystis nidulans mutants resistant to aromatic amino acid analogues.
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Three classes of mutants of Anacystis nidulans were selected on the basis of resistance to fluorophenylalanine and 2-amino-3-phenylbutanoic acid. The most frequent type exhibited DAHP synthetase (7-phospho-2-keto-3-deoxy-D-arabino-heptonate-D-erythrose-4-phosphate-lyase [pyruvate phosphorylating], EC 4.1.2.15) activity identical to that of the parental strain. The second type was characterized by extremely low levels of the activity. The third type had a DAHP synthetase showing decreased sensitivity to inhibition by L-tyrosine. The enzyme was purified 140-fold from wild-type and feedback-insensitive strains, and the kinetics of the reaction was examined. The activity of the wild-type enzyme was inhibited 75% in the presence of 2.0 X 10-3 M tyrosine, and the altered enzyme was inhibited 10%. The following apparent constants were obtained from kinetic studies with partially purified wild-type enzyme: S0.5 for D-erythrose-4-phophate equal to 7.1 X 10-4 M; S0.5 for phosphoenolpyruvate equal to 1.4 X 10-4 M. Inhibition by tyrosine was mixed with respect to binding of both D-erythrose-4-phosphate and phosphoenolpyruvate. In addition, tyrosine promoted cooperative interactions in the binding of phosphoenolpyruvate. For the altered enzyme the following apparent constants were obtained: S0.5 for D-erythrose-4-phosphate equal to 7.1 X 10-4 M; S0.5 for phosphoenolpyruvate equal to 2.9 X 10-4 M. Inhibition by tyrosine was mixed with respect to D-erythrose-4-phosphate and competitive with respect to phosphoenolpyruvate. Tyrosine did not promote cooperative effects in the binding of phosphoenolpyruvate to the altered enzyme. (+info)