title: Functional characterization of AlgL, an alginate lyase from Pseudomonas aeruginosa.
abstract: Alginate lyase (AlgL) catalyzes the cleavage of the polysaccharide alginate through a β-elimination reaction. In Pseudomonas aeruginosa, algL is part of the alginate biosynthetic operon, and although it is required for alginate biosynthesis, it is not clear why. Steady-state kinetic studies were performed to characterize its substrate specificity and revealed that AlgL operates preferentially on nonacetylated alginate or its precursor mannuronan. Mature alginate is secreted as a partially acetylated polysaccharide, so this observation is consistent with suggestions that AlgL serves to degrade mislocalized alginate that is trapped in the periplasmic space. The k(cat)/K(m) for the reaction increased linearly with the number of residues in the substrate, from 2.1 × 10(5) M(-1) s(-1) for the substrate containing 16 residues to 7.9 × 10(6) M(-1) s(-1) for the substrate with 280 residues. Over the same substrate size range, k(cat) varied between 10 and 30 s(-1). The variation in k(cat)/K(m) with substrate length suggests that AlgL operates in a processive manner. AlgL displayed a surprising lack of stereospecificity, in that it was able to catalyze cleavage adjacent to either mannuronate or guluronate residues in alginate. Thus, the enzyme is able to remove the C5 proton from both mannuronate and guluronate, which are C5 epimers. Exhaustive digestion of alginate by AlgL generated dimeric and trimeric products, which were characterized by (1)H nuclear magnetic resonance spectroscopy and mass spectrometry. Rapid-mixing chemical quench studies revealed that there was no lag in dimer or trimer production, indicating that AlgL operates as an exopolysaccharide lyase.
authors.affiliation: Department of Biochemistry, University of Missouri, Columbia, MO 65211, USA.
authors.forename: Emma K
authors.initials: EK
authors.lastname: Farrell
authors.forename: Peter A
authors.initials: PA
authors.lastname: Tipton
mesh_terms: D000464:Alginates; D020723:Glucuronic Acid / Q000096:biosynthesis; D006603:Hexuronic Acids; D007700:Kinetics; D011133:Polysaccharide-Lyases / Q000302:isolation & purification / Q000378:metabolism*; D011550:Pseudomonas aeruginosa / Q000201:enzymology; D013379:Substrate Specificity
chemical_list: D000464:Alginates; D006603:Hexuronic Acids; D020723:Glucuronic Acid; D011133:Polysaccharide-Lyases; C016158:poly(beta-D-mannuronate) lyase