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| PURIFICATION AND CHARACTERIZATION OF PHOSPHOMANNOSE ISOMERASE-GUANOSINE DIPHOSPHO-D-MANNOSE PYROPHOSPHORYLASE - A BIFUNCTIONAL ENZYME IN THE ALGINATE BIOSYNTHETIC-PATHWAY OF PSEUDOMONAS-AERUGINOSA |
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| Author(s): SHINABARGER D, BERRY A, MAY TB, ROTHMEL R, FIALHO A, CHAKRABARTY AM |
| Source: JOURNAL OF BIOLOGICAL CHEMISTRY Volume: 266 Issue: 4 Pages: 2080-2088 Published: FEB 5 1991 |
| Times Cited: 88 References: 39 |
| 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 K(m) for D-mannose 6-phosphate was 3.03 mM, and the V(max) was 830 nmol/min/mg of enzyme. For the GMP forward reaction, apparent K(m) values of 20.5-mu-M and 29.5-mu-M for D-mannose 1-phosphate and GTP, respectively, were obtained from double reciprocal plots. The GMP forward reaction V(max) (5,680 nmol)/min/mg of enzyme) was comparable to the reverse reaction V(max) (5,170 nmol/min/mg of enzyme), and the apparent K(m) for GDP-D-mannose was determined to be 14.2-mu-M. 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. |
| Document Type: Article |
| Language: English |
Addresses:
1. UNIV ILLINOIS, COLL MED, DEPT MICROBIOL & IMMUNOL, 835 S WOLCOTT AVE, CHICAGO, IL 60612 USA |
| Publisher: AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, 9650 ROCKVILLE PIKE, BETHESDA, MD 20814 |
| Subject Category: Biochemistry & Molecular Biology |
| IDS Number: EV515 |
| ISSN: 0021-9258 |
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| | | | | | | | | Record from Web of Science® | | | | | | | | | |