Pseudomonas aeruginosa C5-mannuronan epimerase: steady-state kinetics and characterization of the product.
Alginate is a major constituent of mature biofilms produced by Pseudomonas aeruginosa. The penultimate step in the biosynthesis of alginate is the conversion of some beta-D-mannuronate residues in the polymeric substrate polymannuronan to alpha-L-guluronate residues in a reaction catalyzed by C5-mannuronan epimerase. Specificity studies conducted with size-fractionated oligomannuronates revealed that the minimal substrate contained nine monosaccharide residues. The maximum velocity of the reaction increased from 0.0018 to 0.0218 s(-1) as the substrate size increased from 10 to 20 residues, and no additional increase in kcat was observed for substrates up to 100 residues in length. The Km decreased from 80 microM for a substrate containing fewer than 15 residues to 4 microM for a substrate containing more than 100 residues. In contrast to C5-mannuronan epimerases that have been characterized in other bacterial species, P. aeruginosa C5-mannuronan epimerase does not require Ca2+ for activity, and the Ca2+-alginate complex is not a substrate for the enzyme. Analysis of the purified, active enzyme by inductively coupled plasma-emission spectroscopy revealed that no metals were present in the protein. The pH dependence of the kinetic parameters revealed that three residues on the enzyme which all have a pKa of approximately 7.6 must be protonated for catalysis to occur. The composition of the polymeric product of the epimerase reaction was analyzed by 1H NMR spectroscopy, which revealed that tracts of adjacent guluronate residues were readily formed. The reaction reached an apparent equilibrium when the guluronate composition of the polymer was 75%.
Jerga
Agoston
A
Department of Biochemistry, University of Missouri, Columbia, Missouri 65211, USA.
Raychaudhuri
Aniruddha
A
Tipton
Peter A
PA
D000464:Alginates; D002118:Calcium / Q000378:metabolism; D002238:Carbohydrate Epimerases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D020723:Glucuronic Acid / Q000096:biosynthesis; D006603:Hexuronic Acids / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008670:Metals / Q000378:metabolism; D011550:Pseudomonas aeruginosa / Q000201:enzymology* / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D013379:Substrate Specificity
D000464:Alginates; D006603:Hexuronic Acids; D008670:Metals; D011994:Recombinant Proteins; C007896:guluronic acid; D020723:Glucuronic Acid; D002238:Carbohydrate Epimerases; C086519:mannuronan c-5-epimerase; D002118:Calcium