Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:4.1.2.13 (aldolase)
 3,461 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

When neuroinvasive Escherichia coli K1 cells are grown at temperatures below 20 degrees C, they fail to synthesize the alpha-2,8-linked polysialic acid (polySia) capsule. The objective of this study was to use a genetic and biochemical approach to analyse why capsule expression was defective at cold temperatures. The strategy was to construct E.coli K1-derived mutants with defects in activation and degradation of Sia. The inability to degrade Sia because of a defect in the Sia-specific aldolase permitted accurate quantitation of Sia and CMP-Sia. Strains EV5 and EV90 possessed a defective CMP-Sia synthetase and were unable to activate Sia. These mutants were then used to study how synthesis of Sia, CMP-Sia, and the polySia capsule was affected by growth at 15 degrees C. In contrast to wild type strains, the mutants accumulated Sia in considerable quantities (up to 100 nmol mg protein-1) at 37 degrees C. However, no Sia was detected after growth at 15 degrees C. A temperature upshift experiment showed that the intracellular concentration of Sia increased ca. 3-fold within 5-10 min after shift from 15 to 37 degrees C, even in the presence of inhibitors of protein synthesis or transcription initiation. An in vitro assay for Sia synthase showed that Sia was synthesized at 37 degrees C in cell-free extracts from both 37 and 15 degrees C grown cells, but that no synthesis occurred when the same extracts were assayed at 15 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Biosynthesis of the polysialic acid capsule in Escherichia coli K1. Cold inactivation of sialic acid synthase regulates capsule expression below 20 degrees C. 213 86

In Escherichia coli, synthesis of sialic acid is not regulated by allosteric inhibition mediated by cytidine 5'-monophospho-N-acetylneuraminic acid (CMP-NeuNAc). Evidence for the lack of metabolic control by feedback inhibition was demonstrated by measuring the intracellular level of sialic acid and CMP-NeuNAc in mutants defective in sialic acid polymerization and in CMP-NeuNAc synthesis. Polymerization-defective mutants could not synthesize the polysialic acid capsule and accumulated ca. 25-fold more CMP-NeuNAc than the wild type. Mutants unable to activate sialic acid because of a defect in CMP-NeuNAc synthetase accumulated ca. sevenfold more sialic acid than the wild type. An additional threefold increase in sialic acid levels occurred when a mutation resulting in loss of N-acylneuraminate pyruvate-lysase (sialic acid aldolase) was introduced into the CMP-NeuNAc synthetase-deficient mutant. The aldolase mutation could not be introduced into the polymerization-defective mutant, suggesting that any further increase in the intracellular CMP-NeuNAc concentration was toxic. These results show that sialic acid aldolase can regulate the intracellular concentration of sialic acid and therefore the concentration of CMP-NeuNAc. We conclude that regulation of aldolase, mediated by sialic acid induction, is necessary not only for dissimilating sialic acid (E.R. Vimr and F. A. Troy, J. Bacteriol. 164:845-853, 1985) but also for modulating the level of metabolic intermediates in the sialic acid pathway. In agreement with this conclusion, an increase in the intracellular sialic acid concentration was correlated with an increase in aldolase activity. Direct evidence for the central role of aldolase in regulating the metabolic flux of sialic adid in E. coli was provided by the finding that exogenous radiolabeled sialic acid was specifically incorporated into sialyl polymer in aldolase-negative strain but not in the wild type.
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PMID:Regulation of sialic acid metabolism in Escherichia coli: role of N-acylneuraminate pyruvate-lyase. 390

Escherichia coli K1 produces a capsular polysaccharide of alpha(2-8) poly-N-acetylneuraminic acid. This polysaccharide is an essential virulence factor of these neuropathogenic bacteria. The genes necessary for the synthesis of neuNAc were localized to a plasmid containing the neuBAC genes of the K1 gene cluster. Cells harboring the neuB+ allele in an aldolase (nanA-) negative background produce neuNAc in vivo. Enzymatic synthesis of neuNAc could be demonstrated in extracts of cells harboring an expression plasmid (pNEUB) containing the neuB gene alone. NeuNAc synthetase was purified to homogeneity from extracts of cells harboring pNEUB. The molecular weight of the purified enzyme is 40 kDa, similar to that predicted by the nucleotide sequence of the neuB gene. The amino terminal sequence of the purified protein matches that predicted by the nucleotide sequence of the neuB gene. NeuNAc synthetase catalyzes the formation of neuNAc as indicated by its coupling to the CMP-neuNAc synthetase reaction. The enzyme condenses manNAc and PEP with the release of phosphate. The E. coli neuNAc synthetase is specific for manNAc and PEP, unlike rat liver enzyme that utilizes N-acetylmannosamine-6-phosphate to form neuNAc-9-PO4. This represents the first report of a purification of a sialic acid synthetase from either a eukaryotic or prokaryotic source to homogeneity. These experiments clearly demonstrate an aldolase-independent sialic acid synthetase activity in E. coli K1.
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PMID:Purification and characterization of the Escherichia coli K1 neuB gene product N-acetylneuraminic acid synthetase. 925 51

Most microorganisms do not produce sialic acid (sialate), and those that do appear to use a biosynthetic mechanism distinct from mammals. Genetic hybrids of nonpathogenic, sialate-negative laboratory Escherichia coli K-12 strains designed for the de novo synthesis of the polysialic acid capsule from E. coli K1 proved useful in elucidating the genetics and biochemistry of capsule biosynthesis. In this article we propose a dynamic model of sialometabolism to investigate the effects of biosynthetic neu (N-acetylneuraminic acid) and catabolic nan (N-acylneuraminate) mutations on the flux of intermediates through the sialate synthetic pathway. Intracellular sialate concentrations were determined by high pH anion exchange chromatography with pulsed amperometric detection. The results indicated that a strain carrying a null defect in the gene encoding polysialyltransferase (neuS) accumulated > 50 times more CMP-sialic acid than the wild type when strains were grown in a minimal medium supplemented with glucose and casamino acids. Metabolic accumulation of CMP-sialic acid depended on a functional sialic acid synthase (neuB), as shown by the inability of a strain lacking this enzyme to accumulate a detectable endogenous sialate pool. The neuB mutant concentrated trace sialate from the medium, indicating its potential value for quantitative analysis of free sialic acids in complex biological samples. The function of the sialate aldolase (encoded by nanA) in limiting intermediate flux through the synthetic pathway was determined by analyzing free sialate accumulation in neuA (CMP-sialic acid synthetase) nanA double mutants. The combined results demonstrate how E. coli avoids a futile cycle in which biosynthetic sialate induces the system for its own degradation and indicate the feasibility of generating sialooligosaccharide precursors through targeted manipulation of sialate metabolism.
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PMID:Redirection of sialic acid metabolism in genetically engineered Escherichia coli. 1144 32

When fed to a beta-galactosidase-negative (lacZ(-)) Escherichia coli strain that was grown on an alternative carbon source (such as glycerol), lactose accumulated intracellularly on induction of the lactose permease. We showed that intracellular lactose was efficiently glycosylated when genes of glycosyltransferase that use lactose as acceptor were expressed. High-cell-density cultivation of lacZ(-) strains that overexpressed the beta 1,3 N acetyl glucosaminyltransferase lgtA gene of Neisseria meningitidis resulted in the synthesis of 6 g x L(-1) of the expected trisaccharide (GlcNAc beta 1-3Gal beta 1-4Glc). When the beta 1,4 galactosyltransferase lgtB gene of N. meningitidis was coexpressed with lgtA, the trisaccharide was further converted to lacto-N-neotetraose (Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc) and lacto-N-neoheaxose with a yield higher than 5 g x L(-1). In a similar way, the nanA(-) E. coli strain that was devoid of NeuAc aldolase activity accumulated NeuAc on induction of the NanT permease and the lacZ(-) nanA(-) strain that overexpressed the N. meningitidis genes of the alpha2,3 sialyltransferase and of the CMP-NeuAc synthase efficiently produced sialyllactose (NeuAc alpha 2-3Gal beta 1-4Glc) from exogenous NeuAc and lactose.
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PMID:A new fermentation process allows large-scale production of human milk oligosaccharides by metabolically engineered bacteria. 1204 46

Three C terminal His6-tagged recombinant microbial CMP-sialic acid synthetases [EC 2.7.7.43] cloned from Neisseria meningitidis group B, Streptococcus agalactiae serotype V, and Escherichia coli K1, respectively, were evaluated for their ability in the synthesis of CMP-sialic acid derivatives in a one-pot two-enzyme system. In this system, N-acetylmannosamine or mannose analogs were condensed with pyruvate, catalyzed by a recombinant sialic acid aldolase [EC 4.1.3.3] cloned from E. coli K12 to provide sialic acid analogs as substrates for the CMP-sialic acid synthetases. The substrate flexibility and the reaction efficiency of the three recombinant CMP-sialic acid synthetases were compared, first by qualitative screening using thin layer chromatography, and then by quantitative analysis using high performance liquid chromatography. The N. meningitidis synthetase was shown to have the highest expression level, the most flexible substrate specificity, and the highest catalytic efficiency among the three synthetases. Finally, eight sugar nucleotides, including cytidine 5'-monophosphate N-acetylneuraminic acid (CMP-Neu5Ac) and its derivatives with substitutions at carbon-5, carbon-8, or carbon-9 of Neu5Ac, were synthesized in a preparative (100-200 mg) scale from their 5- or 6-carbon sugar precursors using the N. meningitidis synthetase and the aldolase.
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PMID:Chemoenzymatic synthesis of CMP-sialic acid derivatives by a one-pot two-enzyme system: comparison of substrate flexibility of three microbial CMP-sialic acid synthetases. 1555 60

We have previously described a microbiological process for the conversion of lactose into 3'sialyllactose and other ganglioside sugars by living Escherichia coli cells expressing the appropriate recombinant glycosyltransferase genes. In this system the activated sialic acid donor (CMP-Neu5Ac) was generated from exogenous sialic acid, which was transported into the cells by the permease NanT. Since sialic acid is an expensive compound, a more economical process has now been developed by genetically engineering E. coli K12 to be capable of generating CMP-Neu5Ac using its own internal metabolism. Mutant strains devoid of Neu5Ac aldolase and of ManNAc kinase were shown to efficiently produce 3'sialyllactose by coexpressing the alpha-2,3-sialyltransferase gene from Neisseria meningitidis with the neuC, neuB and neuACampylobacter jejuni genes encoding N-acetylglucosamine-6-phosphate-epimerase, sialic acid synthase and CMP-Neu5Ac synthetase, respectively. A sialyllactose concentration of 25 g l(-1) was obtained in long-term high cell density culture with a continuous lactose feed. This high concentration and low cost of fermentation medium should make possible to use sialylated oligosaccharides in new fields such as the food industry.
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PMID:Genetic engineering of Escherichia coli for the economical production of sialylated oligosaccharides. 1837 33