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

• Published in: The Journal of biological chemistry Vol. 269; no. 7; pp. 4872 - 4877
• Main Authors: MAY, T. B, SHINABARGER, D, BOYD, A, CHAKRABARTY, A. M
• Format: Journal Article
• Language: English
• Published: Bethesda, MD American Society for Biochemistry and Molecular Biology 18-02-1994
• Subjects: Alginates - metabolism; Amino Acid Sequence; Analytical, structural and metabolic biochemistry; Bacterial Proteins; Biological and medical sciences; Cloning, Molecular; Electrophoresis, Polyacrylamide Gel; Enzymes and enzyme inhibitors; Escherichia coli; Fundamental and applied biological sciences. Psychology; Genes, Bacterial; Kinetics; Mannose-6-Phosphate Isomerase - biosynthesis; Mannose-6-Phosphate Isomerase - isolation & purification; Mannose-6-Phosphate Isomerase - metabolism; Miscellaneous; Models, Biological; Molecular Sequence Data; Multienzyme Complexes - biosynthesis; Multienzyme Complexes - isolation & purification; Multienzyme Complexes - metabolism; Mutagenesis, Site-Directed; Nucleotidyltransferases - biosynthesis; Nucleotidyltransferases - isolation & purification; Nucleotidyltransferases - metabolism; Peptide Fragments - isolation & purification; Phenotype; Plasmids; Pseudomonas aeruginosa; Pseudomonas aeruginosa - enzymology; Pseudomonas aeruginosa - genetics; Recombinant Proteins - biosynthesis; Recombinant Proteins - isolation & purification; Recombinant Proteins - metabolism; Restriction Mapping; Sequence Homology, Amino Acid; Pseudomonadales; Intramolecular oxidoreductases; Enzyme; Site directed mutagenesis; Binding site; Alginates; Pyrophosphorylase; Isomerases; Structure activity relation; Mannose-6-phosphate isomerase; Bacteria; Pseudomonadaceae; Pseudomonas aeruginosa
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(17)37625-1

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.