Enzymatic analysis of uridine diphosphate N-acetyl-d -glucosamine

Seema C. Namboori; David E. Graham

doi:10.1016/j.ab.2008.06.034

Enzymatic analysis of uridine diphosphate N-acetyl-d -glucosamine

Seema C. Namboori, David E. Graham

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25 Scopus citations

Abstract

The Methanococcus maripaludis MMP0352 protein belongs to an oxidoreductase family that has been proposed to catalyze the NAD⁺-dependent oxidation of the 3′′ position of uridine diphosphate N-acetyl-d-glucosamine (UDP-GlcNAc), forming a 3-hexulose sugar nucleotide. The heterologously expressed MMP0352 protein was purified and shown to efficiently catalyze UDP-GlcNAc oxidation, forming one NADH equivalent. This enzyme was used to develop a fixed endpoint fluorometric method to analyze UDP-GlcNAc. The enzyme is highly specific for this acetamido sugar nucleotide, and the procedure had a detection limit of 0.2 μM UDP-GlcNAc in a 1-ml sample. Using the method of standard addition, UDP-GlcNAc concentrations were measured in deproteinized extracts of Escherichia coli, Saccharomyces cerevisiae, and HeLa carcinoma cells. Equivalent concentrations were determined by both enzymatic and chromatographic analyses, validating this method. This procedure can be adapted for the high-throughput analysis of changes in cellular UDP-GlcNAc concentrations in time series experiments or inhibitor screens.

Original language	English
Pages (from-to)	94-100
Number of pages	7
Journal	Analytical Biochemistry
Volume	381
Issue number	1
DOIs	https://doi.org/10.1016/j.ab.2008.06.034
State	Published - Oct 1 2008
Externally published	Yes

Funding

This work was supported in part by Public Health Service grant AI064444 from the National Institute of Allergy and Infectious Diseases and by the Petroleum Research Foundation (44382-G4). We thank Mehdi Moini and Lara Mahal for helpful discussions and also thank Susan Anderson for the gift of HeLa cells.

Funders	Funder number
Petroleum Research Foundation	44382-G4
National Institute of Allergy and Infectious Diseases	R21AI064444
U.S. Public Health Service	AI064444

Keywords

Endpoint assay
Fluorescence
Oxidoreductase
Posttranslational glycosylation
Sugar nucleotide
UDP-N-acetyl-d-glucosamine

UN SDGs

This output contributes to the following UN Sustainable Development Goals (SDGs)

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10.1016/j.ab.2008.06.034

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abstract = "The Methanococcus maripaludis MMP0352 protein belongs to an oxidoreductase family that has been proposed to catalyze the NAD+-dependent oxidation of the 3′′ position of uridine diphosphate N-acetyl-d-glucosamine (UDP-GlcNAc), forming a 3-hexulose sugar nucleotide. The heterologously expressed MMP0352 protein was purified and shown to efficiently catalyze UDP-GlcNAc oxidation, forming one NADH equivalent. This enzyme was used to develop a fixed endpoint fluorometric method to analyze UDP-GlcNAc. The enzyme is highly specific for this acetamido sugar nucleotide, and the procedure had a detection limit of 0.2 μM UDP-GlcNAc in a 1-ml sample. Using the method of standard addition, UDP-GlcNAc concentrations were measured in deproteinized extracts of Escherichia coli, Saccharomyces cerevisiae, and HeLa carcinoma cells. Equivalent concentrations were determined by both enzymatic and chromatographic analyses, validating this method. This procedure can be adapted for the high-throughput analysis of changes in cellular UDP-GlcNAc concentrations in time series experiments or inhibitor screens.",

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AB - The Methanococcus maripaludis MMP0352 protein belongs to an oxidoreductase family that has been proposed to catalyze the NAD+-dependent oxidation of the 3′′ position of uridine diphosphate N-acetyl-d-glucosamine (UDP-GlcNAc), forming a 3-hexulose sugar nucleotide. The heterologously expressed MMP0352 protein was purified and shown to efficiently catalyze UDP-GlcNAc oxidation, forming one NADH equivalent. This enzyme was used to develop a fixed endpoint fluorometric method to analyze UDP-GlcNAc. The enzyme is highly specific for this acetamido sugar nucleotide, and the procedure had a detection limit of 0.2 μM UDP-GlcNAc in a 1-ml sample. Using the method of standard addition, UDP-GlcNAc concentrations were measured in deproteinized extracts of Escherichia coli, Saccharomyces cerevisiae, and HeLa carcinoma cells. Equivalent concentrations were determined by both enzymatic and chromatographic analyses, validating this method. This procedure can be adapted for the high-throughput analysis of changes in cellular UDP-GlcNAc concentrations in time series experiments or inhibitor screens.

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Enzymatic analysis of uridine diphosphate N-acetyl-d -glucosamine

Abstract

Funding

Keywords

UN SDGs

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