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

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 K , for D-mannOSe 6-phosphate was 3.03 mM, and the V,,, was 830 nmol/min/mg of enzyme.For the GMP forward reaction, apparent K , values of 20.5 p~ and 29.5 p~ for D-mannOSe 1-phosphate and GTP, respectively, were obtained from double reciprocal plots.The GMP forward reaction V,,, (5,680 nmol/ min/mg of enzyme) was comparable to the reverse reaction v,,, (5,170 nmol/min/mg of enzyme), and the apparent K , for GDP-D-mannose was determined to be 14.2 p ~.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 muta-