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Cloning and characterization of phosphomannose isomerase from Sphingomonas chungbukensis DJ77
Sinh Thi Tran1, Dung Tien Le, Young-Chang Kim
1Deparment of Biochemistry, Chungbuk National University, Cheongju 361-763, Korea.
Abstract:
Phosphomannose isomerase (PMI) catalyzes the interconversion of fructose-6-phosphate and mannose-6-phosphate in the extracellular polysaccharide (EPS) synthesis pathway. The gene encoding PMI in Sphingomonas chungbukensis DJ77 was cloned and expressed in E. coli. The pmi gene is 1,410 nucleotides long and the deduced amino acid sequence shares high homology with other bifunctional proteins that possess both PMI and GDP-mannose pyrophosphorylase (GMP) activities. The sequence analysis of PMI revealed two domains with three conserved motifs: a GMP domain at the N-terminus and a PMI domain at the C-terminus. Enzyme assays using the PMI protein confirmed its bifunctional activity. Both activities required divalent metal ions such as Co(2+), Ca(2+), Mg(2+), Ni(2+) or Zn(2+). Of these ions, Co(2+) was found to be the most effective activator of PMI. GDP-D-mannose was found to inhibit the PMI activity, suggesting feedback regulation of this pathway.
Insights
Phosphomannose isomerase (PMI) from Sphingomonas chungbukensis DJ77 is bifunctional, possessing both PMI and GDP-mannose pyrophosphorylase (GMP) activities. This enzyme is crucial for extracellular polysaccharide synthesis and shows feedback regulation.
Area of Science:
- Biochemistry
- Molecular Biology
- Microbiology
Background:
- Extracellular polysaccharide (EPS) synthesis is vital for microbial communities.
- Phosphomannose isomerase (PMI) plays a key role in the EPS pathway.
- Understanding PMI function is crucial for metabolic engineering and biotechnology.
Purpose of the Study:
- To clone and characterize the phosphomannose isomerase (PMI) gene from Sphingomonas chungbukensis DJ77.
- To investigate the enzymatic activities and regulatory mechanisms of PMI.
- To explore the potential of PMI in biotechnological applications.
Main Methods:
- Gene cloning and expression in E. coli.
- Bioinformatic analysis of the deduced amino acid sequence.
- Enzyme activity assays to confirm bifunctional PMI and GDP-mannose pyrophosphorylase (GMP) activities.
- Metal ion dependency and inhibition studies.
Main Results:
- The pmi gene from S. chungbukensis DJ77 was successfully cloned and expressed.
- The deduced amino acid sequence revealed homology to bifunctional PMI/GMP proteins with distinct N-terminal GMP and C-terminal PMI domains.
- Enzyme assays confirmed bifunctional activity, with optimal activation by Co(2+).
- GDP-D-mannose inhibited PMI activity, indicating feedback regulation.
Conclusions:
- Sphingomonas chungbukensis DJ77 possesses a bifunctional phosphomannose isomerase with GDP-mannose pyrophosphorylase activity.
- The enzyme's activity is dependent on divalent metal ions and subject to feedback inhibition.
- This characterization provides insights into EPS synthesis regulation and potential for enzyme engineering.
