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

J Biol Chem. 1991 Feb 5;266(4):2080-8.

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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Alginates / metabolism
  • Amino Acid Sequence
  • Base Sequence
  • Chromatography, Gel
  • Chromatography, Ion Exchange
  • Genes, Bacterial
  • Mannose-6-Phosphate Isomerase / genetics
  • Mannose-6-Phosphate Isomerase / isolation & purification
  • Mannose-6-Phosphate Isomerase / metabolism*
  • Molecular Sequence Data
  • Molecular Weight
  • Multienzyme Complexes / genetics
  • Multienzyme Complexes / isolation & purification
  • Multienzyme Complexes / metabolism*
  • Nucleotidyltransferases / genetics
  • Nucleotidyltransferases / isolation & purification
  • Nucleotidyltransferases / metabolism
  • Pseudomonas aeruginosa / enzymology
  • Pseudomonas aeruginosa / genetics

Substances

  • Alginates
  • Multienzyme Complexes
  • Nucleotidyltransferases
  • mannose 1-phosphate guanylyltransferase
  • Mannose-6-Phosphate Isomerase

Associated data

  • GENBANK/M14037
  • GENBANK/S61326
  • GENBANK/S61330
  • GENBANK/S61332
  • GENBANK/S61335
  • GENBANK/S61337
  • GENBANK/S61340
  • GENBANK/S61342
  • GENBANK/S61344
  • GENBANK/S65011