title: Purification and characterization of phosphomannomutase/phosphoglucomutase from Pseudomonas aeruginosa involved in biosynthesis of both alginate and lipopolysaccharide.
abstract: The algC gene from Pseudomonas aeruginosa has been shown to encode phosphomannomutase (PMM), an essential enzyme for biosynthesis of alginate and lipopolysaccharide (LPS). This gene was overexpressed under control of the tac promoter, and the enzyme was purified and its substrate specificity and metal ion effects were characterized. The enzyme was determined to be a monomer with a molecular mass of 50 kDa. The enzyme catalyzed the interconversion of mannose 1-phosphate (M1P) and mannose 6-phosphate, as well as that of glucose 1-phosphate (G1P) and glucose 6-phosphate. The apparent Km values for M1P and G1P were 17 and 22 microM, respectively. On the basis of Kcat/Km ratio, the catalytic efficiency for G1P was about twofold higher than that for M1P. PMM also catalyzed the conversion of ribose 1-phosphate and 2-deoxyglucose 6-phosphate to their corresponding isomers, although activities were much lower. Purified PMM/phosphoglucomutase (PGM) required Mg2+ for maximum activity; Mn2+ was the only other divalent metal that showed some activation. The presence of other divalent metals in addition to Mg2+ in the reaction inhibited the enzymatic activity. PMM and PGM activities could not be detected in nonmucoid algC mutant strain 8858 and in LPS-rough algC mutant strain AK1012, while they were present in the wild-type strains as well as in algC-complemented mutant strains. This evidence suggests that AlgC functions as PMM and PGM in vivo, converting phosphomannose and phosphoglucose in the biosynthesis of both alginate and LPS.
authors.affiliation: Department of Microbiology and Immunology, University of Illinois, College of Medicine, Chicago 60612.
authors.forename: R W
authors.initials: RW
authors.lastname: Ye
authors.forename: N A
authors.initials: NA
authors.lastname: Zielinski
authors.forename: A M
authors.initials: AM
authors.lastname: Chakrabarty
mesh_terms: D000464:Alginates / Q000378:metabolism*; D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D002413:Cations, Divalent; D004492:Edetic Acid / Q000494:pharmacology; D004789:Enzyme Activation; D020723:Glucuronic Acid; D006603:Hexuronic Acids; D007700:Kinetics; D008070:Lipopolysaccharides / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D010733:Phosphoglucomutase / Q000378:metabolism*; D017875:Phosphotransferases (Phosphomutases) / Q000378:metabolism*; D011550:Pseudomonas aeruginosa / Q000201:enzymology*; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
chemical_list: D000464:Alginates; D002413:Cations, Divalent; D006603:Hexuronic Acids; D008070:Lipopolysaccharides; D009838:Oligodeoxyribonucleotides; D020723:Glucuronic Acid; D004492:Edetic Acid; D017875:Phosphotransferases (Phosphomutases); D010733:Phosphoglucomutase; C052799:phosphomannomutase