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Identification of Kinase-substrate Pairs Using High Throughput Screening
Published on: August 29, 2015
Substrate promiscuity of N-acetylhexosamine 1-kinases
1Department of Chemistry, University of California, One Shields Avenue, Davis, CA 95616, USA. yhzli@ucdavis.edu
Molecules (Basel, Switzerland)
|July 30, 2011
Summary
Researchers characterized N-acetylhexosamine 1-kinases (NahKs) from Bifidobacterium species. These enzymes efficiently phosphorylate N-acetylhexosamines and accept various modified substrates, showing potential for carbohydrate synthesis.
Area of Science:
- Biochemistry
- Enzymology
- Microbiology
Background:
- N-Acetylhexosamine 1-kinase (NahK) phosphorylates N-acetylhexosamines using ATP.
- Understanding NahK substrate specificity is crucial for carbohydrate synthesis applications.
Purpose of the Study:
- To clone and characterize two NahKs from Bifidobacterium infantis and Bifidobacterium longum.
- To investigate the substrate specificity of these NahKs towards various N-acetylhexosamine derivatives.
- To develop a novel capillary electrophoresis assay for enzyme activity.
Main Methods:
- Gene cloning and expression in E. coli.
- Enzyme activity assays using a newly developed capillary electrophoresis method.
- Substrate specificity studies with native and modified N-acetylglucosamine and N-acetylgalactosamine.
Main Results:
- Both Bifidobacterium NahKs expressed well in E. coli and tolerated diverse modifications at C2, C6, and C3 positions of N-acetylhexosamines.
- Enzymes showed high activity towards mannose and its derivatives, despite low activity towards glucose and galactose.
- NahKs demonstrated broad substrate acceptance, including various modified N-acetylglucosamine derivatives.
Conclusions:
- The characterized Bifidobacterium NahKs are versatile enzymes with broad substrate specificity.
- These enzymes are promising biocatalysts for the enzymatic and chemoenzymatic synthesis of complex carbohydrates.
- The developed capillary electrophoresis assay provides an efficient method for enzyme characterization.
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