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Nucleoside diphosphate kinase from Myxococcus xanthus. II. Biochemical characterization
J Muñoz-Dorado1, S Inouye, M Inouye
1Department of Biochemistry, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, Piscataway 08854-5635.
The Journal of Biological Chemistry
|February 15, 1990
Summary
The Myxococcus xanthus 16-kDa GTP-binding protein was cloned and overexpressed in E. coli. This protein functions as a nucleoside diphosphate kinase, catalyzing reversible phosphorylation reactions.
Area of Science:
- Molecular Biology
- Enzymology
- Bacterial Genetics
Background:
- The 16-kDa GTP-binding protein from Myxococcus xanthus plays a role in cellular processes.
- Understanding its function requires detailed biochemical characterization.
Purpose of the Study:
- To clone and express the gene encoding the 16-kDa GTP-binding protein.
- To purify and characterize the protein's enzymatic activity.
- To determine the protein's function.
Main Methods:
- Gene cloning and expression in Escherichia coli.
- Protein purification using a four-step procedure.
- Gel filtration for molecular weight determination.
- Nucleotide binding and hydrolysis assays.
- Enzyme kinetics studies.
Main Results:
- The gene was successfully cloned and expressed, leading to overproduction of the protein.
- Purified protein exists as a trimer (apparent MW 50,000).
- The protein binds and hydrolyzes nucleoside triphosphates (NTPs) and can transfer phosphate groups.
- Enzymatic activity demonstrated is characteristic of nucleoside diphosphate kinase.
Conclusions:
- The 16-kDa GTP-binding protein from M. xanthus is identified as a nucleoside diphosphate kinase.
- The protein exhibits broad substrate specificity for nucleoside diphosphates and triphosphates.
- The phosphorylation and dephosphorylation reactions proceed via a ping-pong mechanism.