Identification of amino acid residues involved in the activity of phosphomannose isomerase-guanosine 5'-diphospho-D-mannose pyrophosphorylase. A bifunctional enzyme in the alginate biosynthetic pathway of Pseudomonas aeruginosa.
Phosphomannose isomerase-guanosine 5'-diphospho-D-mannose pyrophosphorylase (PMI-GMP), which is encoded by the algA gene, catalyzes two noncontiguous steps in the alginate biosynthetic pathway of Pseudomonas aeruginosa; the isomerization of D-fructose 6-phosphate to D-mannose 6-phosphate and the synthesis of GDP-D-mannose and PPi from GTP and D-mannose 1-phosphate. Amino acids that are required for the GMP enzyme activity were identified through site-directed mutagenesis of the algA gene. Mutation of Lys-175 to arginine, glutamine, or glutamate produced an enzyme whose Km for D-mannose 1-phosphate was 470-3,200-fold greater than that measured for the wild type enzyme. In addition, these mutant enzymes had a lower Vmax for the GMP activity as compared with the wild type PMI-GMP. These results indicate that Lys-175 is primarily involved in the binding of the substrate D-mannose 1-phosphate, although it is likely that other residues are required for the specificity of binding. Mutation of Arg-19 to glutamine, histidine, or leucine resulted in a 2-fold lower Vmax for the GMP enzyme activity and a 4-7-fold increase in the Km for GTP as compared with the wild type enzyme. Thus, it appears that Arg-19 functions in the binding of GTP. In addition, chymotryptic digestion of PMI-GMP showed that the carboxyl terminus is critical for PMI activity but not for GMP activity. Taken together, these results support the hypothesis that the bifunctional PMI-GMP protein is composed of two independent enzymatic domains.
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Department of Microbiology and Immunology, University of Illinois College of Medicine, Chicago 60612.
Shinabarger
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Boyd
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Chakrabarty
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D000464:Alginates / Q000378:metabolism*; D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D005798:Genes, Bacterial; D007700:Kinetics; D008359:Mannose-6-Phosphate Isomerase / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D008954:Models, Biological; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D009713:Nucleotidyltransferases / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D010446:Peptide Fragments / Q000302:isolation & purification; D010641:Phenotype; D010957:Plasmids; D011550:Pseudomonas aeruginosa / Q000201:enzymology* / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D017386:Sequence Homology, Amino Acid
D000464:Alginates; D001426:Bacterial Proteins; D009097:Multienzyme Complexes; D010446:Peptide Fragments; D011994:Recombinant Proteins; C080207:phosphomannose isomerase-guanosine diphospho-D-mannose pyrophosphorylase; D009713:Nucleotidyltransferases; D008359:Mannose-6-Phosphate Isomerase