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Stephan Hinderlich - One of the best experts on this subject based on the ideXlab platform.
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udp glcnac 2 epimerase mannac kinase gne a master regulator of sialic acid synthesis
Topics in Current Chemistry, 2013Co-Authors: Stephan Hinderlich, Tal Yardeni, Rüdiger Horstkorte, Wenke Weidemann, Marjan HuizingAbstract:UDP-N-Acetylglucosamine 2-epimerase/N-acetylmannosamine kinase is the key enzyme of sialic acid biosynthesis in vertebrates. It catalyzes the first two steps of the cytosolic formation of CMP-N-acetylneuraminic acid from UDP-N-Acetylglucosamine. In this review we give an overview of structure, biochemistry, and genetics of the bifunctional enzyme and its complex regulation. Furthermore, we will focus on diseases related to UDP-N-Acetylglucosamine 2-epimerase/N-acetylmannosamine kinase.
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the homozygous m712t mutation of udp n Acetylglucosamine 2 epimerase n acetylmannosamine kinase results in reduced enzyme activities but not in altered overall cellular sialylation in hereditary inclusion body myopathy
FEBS Letters, 2004Co-Authors: Stephan Hinderlich, Rüdiger Horstkorte, Kevin J. Yarema, Lars R. Mantey, Ilan Salama, Iris Eisenberg, Tamara Potikha, Zohar Argov, Menachem Sadeh, Werner ReutterAbstract:Hereditary inclusion body myopathy (HIBM) is a neuromuscular disorder, caused by mutations in UDP-N-Acetylglucosamine 2-epimerase/N-acetylmannosamine kinase, the key enzyme of sialic acid biosynthesis. In Middle Eastern patients a single homozygous mutation occurs, converting methionine-712 to threonine. Recombinant expression of the mutated enzyme revealed slightly reduced N-acetylmannosamine kinase activity, in agreement with the localization of the mutation within the kinase domain. B lymphoblastoid cell lines derived from patients expressing the mutated enzyme also display reduced UDP-N-Acetylglucosamine 2-epimerase activity. Nevertheless, no reduced cellular sialylation was found in those cells by colorimetric assays and lectin analysis, indicating that HIBM is not directly caused by an altered overall expression of sialic acids.
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Efficient biochemical engineering of cellular sialic acids using an unphysiological sialic acid precursor in cells lacking UDP-N-Acetylglucosamine 2-epimerase.
FEBS Letters, 2001Co-Authors: Lars R. Mantey, Michael Pawlita, Werner Reutter, Oliver T Keppler, Stephan HinderlichAbstract:Sialic acids comprise a family of terminal sugars essential for a variety of biological recognition systems. N-Propanoylmannosamine, an unphysiological sialic acid precursor, is taken up and metabolized by mammalian cells resulting in oligosaccharide-bound N-propanoylneuraminic acid. N-Propanoylmannosamine, applied to endogenously hyposialylated subclones of the myeloid leukemia HL60 and of the B-cell lymphoma BJA-B, both deficient in UDP-N-Acetylglucosamine 2-epimerase, is efficiently metabolized to CMP-N-propanoylneuraminic acid resulting in up to 85% of glycoconjugate-associated sialic acids being unphysiological N-propanoylneuraminic acid. Thus, UDP-N-Acetylglucosamine 2-epimerase-deficient cell lines provide an important experimental progress in engineering cells to display an almost homogeneous population of defined, structurally altered sialic acids.
William M. Canfield - One of the best experts on this subject based on the ideXlab platform.
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bovine udp n Acetylglucosamine lysosomal enzyme n Acetylglucosamine 1 phosphotransferase i purification and subunit structure
Journal of Biological Chemistry, 1996Co-Authors: Ming Bao, J L Booth, B J Elmendorf, William M. CanfieldAbstract:UDP-N-Acetylglucosamine:lysosomal-enzyme N-Acetylglucosamine-1-phosphotransferase (GlcNAc-phosphotransferase) catalyzes the initial step in the synthesis of the mannose 6-phosphate determinant required for efficient intracellular targeting of newly synthesized lysosomal hydrolases to the lysosome. The enzyme was partially purified approximately 30,000-fold by chromatography of solubilized membrane proteins from lactating bovine mammary glands on DEAE-Sepharose, reactive green 19-agarose, and Superose 6. The partially purified enzyme was used to generate a panel of murine monoclonal antibodies. The anti-GlcNAc-phosphotransferase monoclonal antibody PT18 was coupled to a solid support and used to immunopurify the enzyme approximately 480,000-fold to apparent homogeneity with an overall yield of 29%. The purified enzyme has a specific activity of 10-12 micromol of GlcNAc phosphate transferred per h/mg using 100 mM alpha-methylmannoside as acceptor. The subunit structure of the enzyme was determined using a combination of analytical gel filtration chromatography, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and amino-terminal sequencing. The data indicate that bovine GlcNAc-phosphotransferase is a 540,000-Da complex composed of disulfide-linked homodimers of 166,000- and 51,000-Da subunits and two identical, noncovalently associated 56,000-Da subunits.
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bovine udp n Acetylglucosamine lysosomal enzyme n Acetylglucosamine 1 phosphotransferase i purification and subunit structure
Journal of Biological Chemistry, 1996Co-Authors: J L Booth, B J Elmendorf, William M. CanfieldAbstract:Abstract UDP-N-Acetylglucosamine:lysosomal-enzyme N-Acetylglucosamine-1-phosphotransferase (GlcNAc-phosphotransferase) catalyzes the initial step in the synthesis of the mannose 6-phosphate determinant required for efficient intracellular targeting of newly synthesized lysosomal hydrolases to the lysosome. The enzyme was partially purified ∼30,000-fold by chromatography of solubilized membrane proteins from lactating bovine mammary glands on DEAE-Sepharose, reactive green 19-agarose, and Superose 6. The partially purified enzyme was used to generate a panel of murine monoclonal antibodies. The anti-GlcNAc-phosphotransferase monoclonal antibody PT18 was coupled to a solid support and used to immunopurify the enzyme ∼480,000-fold to apparent homogeneity with an overall yield of 29%. The purified enzyme has a specific activity of 10-12 μmol of GlcNAc phosphate transferred per h/mg using 100 mM α-methylmannoside as acceptor. The subunit structure of the enzyme was determined using a combination of analytical gel filtration chromatography, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and amino-terminal sequencing. The data indicate that bovine GlcNAc-phosphotransferase is a 540,000-Da complex composed of disulfide-linked homodimers of 166,000- and 51,000-Da subunits and two identical, noncovalently associated 56,000-Da subunits.
Alan D Grund - One of the best experts on this subject based on the ideXlab platform.
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engineering a new pathway for n Acetylglucosamine production coupling a catabolic enzyme glucosamine 6 phosphate deaminase with a biosynthetic enzyme glucosamine 6 phosphate n acetyltransferase
Enzyme and Microbial Technology, 2006Co-Authors: Mingde Deng, Sarah L Wassink, Alan D GrundAbstract:A metabolic pathway for high level production of N-Acetylglucosamine has been engineered in Escherichia coli by overexpressing E. coli glucosamine synthase (GlmS) and Saccharomyces cerevisiae glucosamine-6-phosphate acetyltransferase (GNA1). GlmS catalyzes the synthesis of glucosamine-6-phosphate from fructose-6-phosphate and glutamine. GNA1 converts glucosamine-6-phosphate into N-Acetylglucosamine, which is dephosphorylated and secreted into the growth medium. In the present work, E. coli glucosamine-6-phosphate deaminase (NagB) was evaluated as an alternative to GlmS for the production of glucosamine and N-Acetylglucosamine. NagB is a catabolic enzyme that converts glucosamine-6-phosphate to fructose-6-phosphate and ammonia. The reverse biosynthetic reaction forming glucosamine-6-phosphate is kinetically unfavorable. In a glmS deletion strain requiring glucosamine supplement to survive and grow, NagB overexpression resulted in the synthesis of glucosamine-6-phosphate. This supported cell growth, but little or no glucosamine accumulated in the medium. Overexpression of both NagB and GNA1 resulted in production of N-Acetylglucosamine at levels comparable to strains overexpressing both GlmS and GNA1. This indicates that the overexpression of GNA1 played a critical role in determining the direction and efficiency of the reaction catalyzed by NagB. These data demonstrate that a catabolic enzyme can be utilized in a biosynthetic pathway by coupling with an efficient downstream reaction.
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metabolic engineering of escherichia coli for industrial production of glucosamine and n Acetylglucosamine
Metabolic Engineering, 2005Co-Authors: Mingde Deng, Sarah L Wassink, Alan D Grund, David K Severson, Richard P Burlingame, Alan Berry, Jeffrey A Running, Candice A Kunesh, Linsheng Song, Thomas A JerrellAbstract:Abstract Glucosamine and N -Acetylglucosamine are currently produced by extraction and acid hydrolysis of chitin from shellfish waste. Production could be limited by the amount of raw material available and the product potentially carries the risk of shellfish protein contamination. Escherichia coli was modified by metabolic engineering to develop a fermentation process. Over-expression of glucosamine synthase (GlmS) and inactivation of catabolic genes increased glucosamine production by 15 fold, reaching 60 mg l −1 . Since GlmS is strongly inhibited by glucosamine-6-P, GlmS variants were generated via error-prone PCR and screened. Over-expression of an improved enzyme led to a glucosamine titer of 17 g l −1 . Rapid degradation of glucosamine and inhibitory effects of glucosamine and its degradation products on host cells limited further improvement. An alternative fermentation product, N -Acetylglucosamine, is stable, non-inhibitory to the host and readily hydrolyzed to glucosamine under acidic conditions. Therefore, the glucosamine pathway was extended to N -Acetylglucosamine by over-expressing a heterologous glucosamine-6-P N -acetyltransferase. Using a simple and low-cost fermentation process developed for this strain, over 110 g l −1 of N -Acetylglucosamine was produced.
Mingde Deng - One of the best experts on this subject based on the ideXlab platform.
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engineering a new pathway for n Acetylglucosamine production coupling a catabolic enzyme glucosamine 6 phosphate deaminase with a biosynthetic enzyme glucosamine 6 phosphate n acetyltransferase
Enzyme and Microbial Technology, 2006Co-Authors: Mingde Deng, Sarah L Wassink, Alan D GrundAbstract:A metabolic pathway for high level production of N-Acetylglucosamine has been engineered in Escherichia coli by overexpressing E. coli glucosamine synthase (GlmS) and Saccharomyces cerevisiae glucosamine-6-phosphate acetyltransferase (GNA1). GlmS catalyzes the synthesis of glucosamine-6-phosphate from fructose-6-phosphate and glutamine. GNA1 converts glucosamine-6-phosphate into N-Acetylglucosamine, which is dephosphorylated and secreted into the growth medium. In the present work, E. coli glucosamine-6-phosphate deaminase (NagB) was evaluated as an alternative to GlmS for the production of glucosamine and N-Acetylglucosamine. NagB is a catabolic enzyme that converts glucosamine-6-phosphate to fructose-6-phosphate and ammonia. The reverse biosynthetic reaction forming glucosamine-6-phosphate is kinetically unfavorable. In a glmS deletion strain requiring glucosamine supplement to survive and grow, NagB overexpression resulted in the synthesis of glucosamine-6-phosphate. This supported cell growth, but little or no glucosamine accumulated in the medium. Overexpression of both NagB and GNA1 resulted in production of N-Acetylglucosamine at levels comparable to strains overexpressing both GlmS and GNA1. This indicates that the overexpression of GNA1 played a critical role in determining the direction and efficiency of the reaction catalyzed by NagB. These data demonstrate that a catabolic enzyme can be utilized in a biosynthetic pathway by coupling with an efficient downstream reaction.
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metabolic engineering of escherichia coli for industrial production of glucosamine and n Acetylglucosamine
Metabolic Engineering, 2005Co-Authors: Mingde Deng, Sarah L Wassink, Alan D Grund, David K Severson, Richard P Burlingame, Alan Berry, Jeffrey A Running, Candice A Kunesh, Linsheng Song, Thomas A JerrellAbstract:Abstract Glucosamine and N -Acetylglucosamine are currently produced by extraction and acid hydrolysis of chitin from shellfish waste. Production could be limited by the amount of raw material available and the product potentially carries the risk of shellfish protein contamination. Escherichia coli was modified by metabolic engineering to develop a fermentation process. Over-expression of glucosamine synthase (GlmS) and inactivation of catabolic genes increased glucosamine production by 15 fold, reaching 60 mg l −1 . Since GlmS is strongly inhibited by glucosamine-6-P, GlmS variants were generated via error-prone PCR and screened. Over-expression of an improved enzyme led to a glucosamine titer of 17 g l −1 . Rapid degradation of glucosamine and inhibitory effects of glucosamine and its degradation products on host cells limited further improvement. An alternative fermentation product, N -Acetylglucosamine, is stable, non-inhibitory to the host and readily hydrolyzed to glucosamine under acidic conditions. Therefore, the glucosamine pathway was extended to N -Acetylglucosamine by over-expressing a heterologous glucosamine-6-P N -acetyltransferase. Using a simple and low-cost fermentation process developed for this strain, over 110 g l −1 of N -Acetylglucosamine was produced.
Werner Reutter - One of the best experts on this subject based on the ideXlab platform.
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the homozygous m712t mutation of udp n Acetylglucosamine 2 epimerase n acetylmannosamine kinase results in reduced enzyme activities but not in altered overall cellular sialylation in hereditary inclusion body myopathy
FEBS Letters, 2004Co-Authors: Stephan Hinderlich, Rüdiger Horstkorte, Kevin J. Yarema, Lars R. Mantey, Ilan Salama, Iris Eisenberg, Tamara Potikha, Zohar Argov, Menachem Sadeh, Werner ReutterAbstract:Hereditary inclusion body myopathy (HIBM) is a neuromuscular disorder, caused by mutations in UDP-N-Acetylglucosamine 2-epimerase/N-acetylmannosamine kinase, the key enzyme of sialic acid biosynthesis. In Middle Eastern patients a single homozygous mutation occurs, converting methionine-712 to threonine. Recombinant expression of the mutated enzyme revealed slightly reduced N-acetylmannosamine kinase activity, in agreement with the localization of the mutation within the kinase domain. B lymphoblastoid cell lines derived from patients expressing the mutated enzyme also display reduced UDP-N-Acetylglucosamine 2-epimerase activity. Nevertheless, no reduced cellular sialylation was found in those cells by colorimetric assays and lectin analysis, indicating that HIBM is not directly caused by an altered overall expression of sialic acids.
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Efficient biochemical engineering of cellular sialic acids using an unphysiological sialic acid precursor in cells lacking UDP-N-Acetylglucosamine 2-epimerase.
FEBS Letters, 2001Co-Authors: Lars R. Mantey, Michael Pawlita, Werner Reutter, Oliver T Keppler, Stephan HinderlichAbstract:Sialic acids comprise a family of terminal sugars essential for a variety of biological recognition systems. N-Propanoylmannosamine, an unphysiological sialic acid precursor, is taken up and metabolized by mammalian cells resulting in oligosaccharide-bound N-propanoylneuraminic acid. N-Propanoylmannosamine, applied to endogenously hyposialylated subclones of the myeloid leukemia HL60 and of the B-cell lymphoma BJA-B, both deficient in UDP-N-Acetylglucosamine 2-epimerase, is efficiently metabolized to CMP-N-propanoylneuraminic acid resulting in up to 85% of glycoconjugate-associated sialic acids being unphysiological N-propanoylneuraminic acid. Thus, UDP-N-Acetylglucosamine 2-epimerase-deficient cell lines provide an important experimental progress in engineering cells to display an almost homogeneous population of defined, structurally altered sialic acids.
