Purification and characterization of guanosine diphospho-D-mannose dehydrogenase. A key enzyme in the biosynthesis of alginate by Pseudomonas aeruginosa.
Alginate-producing Pseudomonas aeruginosa are usually associated with the cystic fibrosis lung environment and contribute to the high mortality rates observed among these patients. The present paper describes the purification and enzymatic properties of guanosine diphospho-D-mannose dehydrogenase (EC 1.1.1.132), a key enzyme in alginate biosynthesis by mucoid P. aeruginosa. The enzyme was overproduced using a plasmid vector containing algD (the gene encoding this enzyme) under control of the tac promoter. It was purified from cell-free lysates by lowering the pH to 5.0, heating the extract to 57.5 degrees C for 10 min, and discarding the protein pellet. The enzyme was selectively precipitated from the supernatant fraction with 45% acetone, resuspended in a 100 mM triethanolamine acetate buffer, pH 7.6, and ultimately purified by Bio-Sil TSK-400 gel filtration chromatography. The subunit molecular weight (Mr 48,000) as well as the N-terminal amino acid sequence corresponded to those predicted from the DNA sequence of algD. The native protein migrated as a hexamer of 290,000 molecular weight upon Bio-Gel A-1.5m gel filtration chromatography. Kinetic analysis demonstrated an apparent Km of 14.9 microM for the substrate GDP-D-mannose and 185 microM for the cofactor NAD+. GDP-D-mannuronic acid was identified as the enzyme reaction product. Several compounds (including GMP, ATP, GDP-D-glucose, and maltose) were found to inhibit enzymatic activity. GMP, the most potent of these inhibitors, exhibited competitive inhibition with an apparent Ki of 22.7 microM. Enzyme activity was also sensitive to the sulfhydryl group modifying agents iodoacetamide and p-hydroxymercuribenzoate. The addition of excess dithiothreitol restored enzyme activity, suggesting a possible involvement of cysteine residues in enzymatic activity.
Roychoudhury
S
S
Department of Microbiology and Immunology, University of Illinois College of Medicine, Chicago 60612.
May
T B
TB
Gill
J F
JF
Singh
S K
SK
Feingold
D S
DS
Chakrabarty
A M
AM
D000464:Alginates / Q000096:biosynthesis* / Q000235:genetics; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*; D002237:Carbohydrate Dehydrogenases / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D006155:Guanosine Diphosphate Mannose / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D011550:Pseudomonas aeruginosa / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism
D000464:Alginates; D001426:Bacterial Proteins; D006155:Guanosine Diphosphate Mannose; D002237:Carbohydrate Dehydrogenases; C020562:GDPmannose dehydrogenase