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: EC:4.1.2.13 (aldolase)
3,461 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Fructose 1,6-diphosphate (FDP) aldolase and 2-keto-3-deoxy-D-gluconate (KDG) aldolase the two key enzymes of Embden-Meyerhof-Parnas (EMP) and the nonphosphorolytic Entner-Doudoroff (ED) pathways respectively, were identified in cell-free extracts of four Aspergillus oryzae strains grown on D-glucose as sole source of carbon. A. oryzae NRRL 3435 gave the highest enzymatic activity for the two enzymes and selected for further studies. Studies on the properties of the two key enzymes indicated that the optimum conditions for the activities of FDP aldolase and KDG aldolases occurred at pH 8.5, 45 degrees C and pH 8.0, 55 degrees C, respectively. Tris-acetate buffer and phosphate buffer showed the highest enzymatic activity for these two enzymes respectively. KDG aldolase was stable at 55 degrees C for 60 minutes however FDP aldolase was found to be less stable above 45 degrees C. On the other hand the two aldolases showed a high degree of stability towards frequent freezing and thawing. Dialysis of the extracts caused a decrease in the enzymatic activity of KDG aldolase, and an increase in FDP aldolase activity. The addition of ethylene diamine tetraacetate to the crude extracts caused an inhibition of KDG aldolase, whileas FDP aldolase was not affected. Addition of MnCl(2), CoSO(4), MgCl(2) and ZnSO(4) to the dialyzed extracts increased the activity of KDG aldolase by 67%, 54%, 61% and 37%, respectively. On the other hand the addition of some metal salts caused an inhibition of FDP aldolase. The results obtained indicate the absence of evidence for the involvement of sulfhydryl groups in the catalytic sites of the two aldolases.
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PMID:Some properties of two aldolases in extracts of Aspergillus oryzae. 1567 61

HpaI, a class II pyruvate-specific aldolase involved in the catabolic pathway of hydroxyphenylacetate, is overexpressed and purified. A previous suggestion that phosphate is involved in proton transfer of pyruvate, based on the crystal structure of the homologous 2-dehydro-3-deoxygalactarate aldolase, is not substantiated from biochemical studies with HpaI. Thus, specific activities of the enzyme for the substrate 4-hydroxy-2-ketopentanoate in sodium HEPES and Tris-acetate buffers are higher than in sodium phosphate buffer. The enzyme also catalyzed the partial reaction of pyruvate proton exchange with an initial rate of 0.77 mmol min(-)(1) mg(-)(1) in phosphate-free buffer, as monitored by nuclear magnetic resonance. Steady-state kinetic analysis shows that the enzyme is also able to catalyze the aldol cleavage of 4-hydroxy-2-ketohexanoate and 3-deoxy-d-manno-oct-2-ulosonic acid (KDO). The enzyme exhibits significant oxaloacetate decarboxylase activity, with a k(cat) value 2.4-fold higher than the corresponding value for the aldol cleavage of 4-hydroxy-2-ketopentanoate. Sodium oxalate, an analogue of the enolate intermediate of the enzyme-catalyzed reaction, is a competitive inhibitor of the enzyme, with a K(i) value of 5.5 microM. Replacement of an active site arginine residue (R70) with alanine by site-specific mutagenesis resulted in an enzyme that lacks both aldolase and decarboxylase activities. The mutant enzyme is also unable to catalyze pyruvate proton exchange. The dissociation constant for pyruvate in the R70A mutant, determined by fluorescence titration, is similar to that of the wild-type enzyme, indicating that pyruvate binding is not affected by this mutation. Together, the results show that R70 influences catalysis in HpaI, particularly at the pyruvate proton exchange step.
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PMID:Purification and biochemical characterization of a pyruvate-specific class II aldolase, HpaI. 1599 99

Under nitrogen (ammonia)-limited continuous culture conditions, the ruminal anaerobe Selenomonas ruminantium was grown at various dilution rates (D). The proportion of the population that was viable increased with D, being 91% at D = 0.5 h. Washed cell suspensions were subjected to long-term nutrient starvation at 39 degrees C. All populations exhibited logarithmic linear declines in viability that were related to the growth rate. Cells grown at D = 0.05, 0.20, and 0.50 lost about 50% viability after 8.1, 4.6, and 3.6 h, respectively. The linear rates of decline in total cell numbers were dramatically less and constant regardless of dilution rate. All major cell constituents declined during starvation, with the rates of decline being greatest with RNA, followed by DNA, carbohydrate, cell dry weight, and protein. The rates of RNA loss increased with cells grown at higher D values, whereas the opposite was observed for rates of carbohydrate losses. The majority of the degraded RNA was not catabolized but was excreted into the suspending buffer. At all D values, S. ruminantium produced mainly lactate and lesser amounts of acetate, propionate, and succinate during growth. With starvation, only small amounts of acetate were produced. Addition of glucose, vitamins, or both to the suspending buffer or starvation in the spent culture medium resulted in greater losses of viability than in buffer alone. Examination of extracts made from starving cells indicated that fructose diphosphate aldolase and lactate dehydrogenase activities remained relatively constant. Both urease and glutamate dehydrogenase activities declined gradually during starvation, whereas glutamine synthetase activity increased slightly. The data indicate that nitrogen (ammonia)-limited S. ruminantium cells have limited survival capacity, but this capacity is greater than that found previously with energy (glucose)-limited cells. Apparently no one cellular constituent serves as a catabolic substrate for endogenous metabolism. Relative to losses in viability, cellular enzymes are stable, indicating that nonviable cells maintain potential metabolic activity and that generalized, nonspecific enzyme degradation is not a major factor contributing to viability loss.
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PMID:Changes in Viability, Cell Composition, and Enzyme Levels During Starvation of Continuously Cultured (Ammonia-Limited) Selenomonas ruminantium. 1634 16

A regulatory system has been described in the obligately phototrophic green alga Chlamydomonas mundana. Cells grown in acetate media are unable to fix carbon dioxide in the light but carry out a photoassimilation of acetate to carbohydrate: cells cultured with carbon dioxide as the sole source of cellular carbon carry out typical green plant photosynthesis. The control appears to take place at the level of the reductive pentose phosphate cycle. The presence of sodium acetate in the medium strongly inhibits formation of ribulose-1.5-diphosphate carboxylase, ribulose-5-phosphate kinase, and one of the 2 fructose-1,6-diphosphate aldolase activities of the cell. Ribose-5-phosphate isomerase is present in higher activity in autotrophic cells. Changes in the levels of triose phosphate dehydrogenase were also noted. The total pigment content of the cell and the photosynthetic electron transport reactions are not altered under different conditions of growth.
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PMID:Regulation of Photosynthetic Capacity in Chlamydomonas mundana. 1665 35

The quantitative estimation of intracellular metabolite concentrations (metabolic profiling) is a prerequisite for a better understanding of biological processes and thus inevitable for the rational improvement of microbial production strains and process design. Since pool sizes of substrates regulate flux through different enzymes, the accurate determination of intracellular metabolite concentrations is necessary to understand in vivo reaction kinetics. Quantification of intracellular concentrations of glycolytic intermediates in Escherichia coli K12 was achieved by using a novel in situ rapid sampling and quenching procedure. A new extraction procedure using buffered hot water was established. By use of simultaneous multi-substrate feeding with various ratios of glucose, fructose and acetate during continuous cultivations several metabolic states were induced. Metabolic flux analysis and the newly developed metabolic profiling procedure were used to determine in vivo enzyme kinetics as exemplified for fructose 1,6-bisphosphate aldolase and citrate synthase.
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PMID:Metabolic profiling of Escherichia coli cultivations: evaluation of extraction and metabolite analysis procedures. 1747 21

The transcriptional regulator SugR from Corynebacterium glutamicum represses genes of the phosphoenolpyruvate-dependent phosphotransferase system (PTS). Growth experiments revealed that the overexpression of sugR not only perturbed the growth of C. glutamicum on the PTS sugars glucose, fructose, and sucrose but also led to a significant growth inhibition on ribose, which is not taken up via the PTS. Chromatin immunoprecipitation combined with DNA microarray analysis and gel retardation experiments were performed to identify further target genes of SugR. Gel retardation analysis confirmed that SugR bound to the promoter regions of genes of the glycolytic enzymes 6-phosphofructokinase (pfkA), fructose-1,6-bisphosphate aldolase (fba), enolase (eno), pyruvate kinase (pyk), and NAD-dependent L-lactate dehydrogenase (ldhA). The deletion of sugR resulted in increased mRNA levels of eno, pyk, and ldhA in acetate medium. Enzyme activity measurements revealed that SugR-mediated repression affects the activities of PfkA, Fba, and LdhA in vivo. As the deletion of sugR led to increased LdhA activity under aerobic and under oxygen deprivation conditions, L-lactate production by C. glutamicum was determined. The overexpression of sugR reduced L-lactate production by about 25%, and sugR deletion increased L-lactate formation under oxygen deprivation conditions by threefold. Thus, SugR functions as a global repressor of genes of the PTS, glycolysis, and fermentative L-lactate dehydrogenase in C. glutamicum.
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PMID:The global repressor SugR controls expression of genes of glycolysis and of the L-lactate dehydrogenase LdhA in Corynebacterium glutamicum. 1884 35

Carnitine is an essential metabolite that enables intracellular transport of fatty acids and acetyl units. Here we show that the yeast Candida albicans can synthesize carnitine de novo, and we identify the 4 genes of the pathway. Null mutants of orf19.4316 (trimethyllysine dioxygenase), orf19.6306 (trimethylaminobutyraldehyde dehydrogenase), and orf19.7131 (butyrobetaine dioxygenase) lacked their respective enzymatic activities and were unable to utilize fatty acids, acetate, or ethanol as a sole carbon source, in accordance with the strict requirement for carnitine-mediated transport under these growth conditions. The second enzyme of carnitine biosynthesis, hydroxytrimethyllysine aldolase, is encoded by orf19.6305, a member of the threonine aldolase (TA) family in C. albicans. A strain lacking orf19.6305 showed strongly reduced growth on fatty acids and was unable to utilize either acetate or ethanol, but TA activity was unaffected. Growth of the null mutants on nonfermentable carbon sources is restored only by carnitine biosynthesis intermediates after the predicted enzymatic block in the pathway, which provides independent evidence for a specific defect in carnitine biosynthesis for each of the mutants. In conclusion, we have genetically characterized a complete carnitine biosynthesis pathway in C. albicans and show that a TA family member is mainly involved in the aldolytic cleavage of hydroxytrimethyllysine in vivo.
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PMID:Identification and characterization of a complete carnitine biosynthesis pathway in Candida albicans. 1928 5

2-Deoxyribose-5-phosphate aldolase (DERA) catalyzes a sequential aldol reaction useful in synthetic chemistry. In this work, the effect of a feeding strategy on the production of a thermophilic DERA was investigated in fed-batch cultures of recombinant Escherichia coli BL21 (pET303-DERA008). The predetermined specific growth rate (micro(set)) was evaluated at 0.20, 0.15, and 0.10 h(-1), respectively. The DERA concentration and volumetric productivity were associated with micro(set). The cells synthesized the enzyme most efficiently at micro(set) = 0.15 h(-1). The maximum enzyme concentration (5.12 g/L) and total volumetric productivity (0.256 g L(-1) h(-1)) obtained were over 10 and five times higher than that from traditional batch cultures. Furthermore, the acetate concentration remained at a relatively low level, less than 0.4 g/L, under this condition which would not inhibit cell growth and target protein expression. Thus, a specific growth rate control strategy has been successfully applied to induce fed-batch cultures for the maximal production of the thermophilic 2-deoxyribose-5-phosphate aldolase.
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PMID:The fed-batch production of a thermophilic 2-deoxyribose-5-phosphate aldolase (DERA) in Escherichia coli by exponential feeding strategy control. 2022 83

Previously, we described the production of N-acetylneuraminic acid (NeuAc) from N-acetylglucosamine (GlcNAc) in a system combining recombinant Escherichia coli expressing GlcNAc 2-epimerase (slr1975), E. coli expressing NeuAc synthetase (neuB), and Corynebacterium ammoniagenes. However, this system was unsuitable for large-scale production because of its complexity and low productivity. To overcome these problems, we constructed a recombinant E. coli simultaneously overexpressing slr1975 and neuB. This recombinant E. coli produced 81mM (25g/L) NeuAc in 22h without the addition of C. ammoniagenes cells. For manufacturing on an industrial scale, it is preferable to use unconcentrated culture broth as the source of enzymes, and therefore, a high-density cell culture is required. An acetate-resistant mutant strain of E. coli (HN0074) was selected as the host strain because of its ability to grow to a high cell density. The NeuAc aldolase gene of E. coli HN0074 was disrupted by homologous recombination yielding E. coli N18-14, which cannot degrade NeuAc. After a 22h reaction with 540mM (120g/L) GlcNAc in a 5L jar fermenter, the culture broth of E. coli N18-14 overexpressing slr1975 and neuB contained 172mM (53g/L) NeuAc.
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PMID:Microbial production of N-acetylneuraminic acid by genetically engineered Escherichia coli. 2097 55

The Archaea harbor many metabolic pathways that differ to previously recognized classical pathways. Glycolysis is carried out by modified versions of the Embden-Meyerhof and Entner-Doudoroff pathways. Thermophilic archaea have recently been found to harbor a bi-functional fructose-1,6-bisphosphate aldolase/phosphatase for gluconeogenesis. A number of novel pentose-degrading pathways have also been recently identified. In terms of anabolic metabolism, a pathway for acetate assimilation, the methylaspartate cycle, and two CO2-fixing pathways, the 3-hydroxypropionate/4-hydroxybutyrate cycle and the dicarboxylate/4-hydroxybutyrate cycle, have been elucidated. As for biosynthetic pathways, recent studies have clarified the enzymes responsible for several steps involved in the biosynthesis of inositol phospholipids, polyamine, coenzyme A, flavin adeninedinucleotide and heme. By examining the presence/absence of homologs of these enzymes on genome sequences, we have found that the majority of these enzymes and pathways are specific to the Archaea.
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PMID:Novel metabolic pathways in Archaea. 2161 76


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