title: Purification and characterization of phosphomannose isomerase-guanosine diphospho-D-mannose pyrophosphorylase. A bifunctional enzyme in the alginate biosynthetic pathway of Pseudomonas aeruginosa.
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.
authors.affiliation: Department of Microbiology and Immunology, University of Illinois College of Medicine, Chicago 60612.
authors.forename: D
authors.initials: D
authors.lastname: Shinabarger
authors.forename: A
authors.initials: A
authors.lastname: Berry
authors.forename: T B
authors.initials: TB
authors.lastname: May
authors.forename: R
authors.initials: R
authors.lastname: Rothmel
authors.forename: A
authors.initials: A
authors.lastname: Fialho
authors.forename: A M
authors.initials: AM
authors.lastname: Chakrabarty
mesh_terms: D000464:Alginates / Q000378:metabolism; D000595:Amino Acid Sequence; D001483:Base Sequence; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D005798:Genes, Bacterial; D008359:Mannose-6-Phosphate Isomerase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009097:Multienzyme Complexes / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D009713:Nucleotidyltransferases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D011550:Pseudomonas aeruginosa / Q000201:enzymology / Q000235:genetics
chemical_list: D000464:Alginates; D009097:Multienzyme Complexes; D009713:Nucleotidyltransferases; C026917:mannose 1-phosphate guanylyltransferase; D008359:Mannose-6-Phosphate Isomerase