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J Biol Chem. 1991 Feb 5;266(4):2080-8.

Purification and characterization of phosphomannose isomerase-guanosine diphospho-D-mannose pyrophosphorylase. A bifunctional enzyme in the alginate biosynthetic pathway of Pseudomonas aeruginosa.

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  • 1Department of Microbiology and Immunology, University of Illinois College of Medicine, Chicago 60612.

Abstract

We report here the purification and characterization of phosphomannose isomerase-guanosine 5'-diphospho-D-mannose pyrophosphorylase, a bifunctional enzyme (PMI-GMP) which catalyzes both the phosphomannose isomerase (PMI) and guanosine 5'-diphospho-D-mannose pyrophosphorylase (GMP) reactions of the Pseudomonas aeruginosa alginate biosynthetic pathway. The PMI and GMP activities co-eluted in the same protein peak through successive fractionation on hydrophobic interaction, ion exchange, and gel filtration chromatography. The purified enzyme migrated as a 56,000 molecular weight protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the native protein migrated as a monomer of 54,000 molecular weight upon gel filtration chromatography. The apparent Km for D-mannose 6-phosphate was 3.03 mM, and the Vmax was 830 nmol/min/mg of enzyme. For the GMP forward reaction, apparent Km values of 20.5 microM and 29.5 microM for D-mannose 1-phosphate and GTP, respectively, were obtained from double reciprocal plots. The GMP forward reaction Vmax (5,680 nmol/min/mg of enzyme) was comparable to the reverse reaction Vmax (5,170 nmol/min/mg of enzyme), and the apparent Km for GDP-D-mannose was determined to be 14.2 microM. Both reactions required Mg2+ activation, but the PMI reaction rate was 4-fold higher with Co2+ as the activator. PMI (but not GMP) activity was sensitive to dithiothreitol, indicating the involvement of disulfide bonds to form a protein structure capable of PMI activity. DNA sequencing of a cloned mutant algA gene from P. aeruginosa revealed that a point mutation at nucleotide 961 greatly decreased the levels of both PMI and GMP in a crude extract.

PMID:
1846611
[PubMed - indexed for MEDLINE]
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