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 non-acetylated 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 kcat/Km for the reaction increased linearly with the number of residues in the substrate, from 2.1×105 M−1s−1 for substrate containing 16 residues to 7.9×106 M−1s−1 for substrate with 280 residues. Over the same substrate size range, kcat varied between 10 s−1 and 30 s−1. The variation in kcat/Km 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 1H NMR 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.

Biological function of a polysaccharide degrading enzyme in the periplasm

“…4). These observations are consistent with findings of Farrell et al 49. which showed that AlgL displayed a surprising lack of stereospecificity to various alginates, further arguing that AlgL functioned exclusively to degrade misguided alginate in the periplasm without being directly involved in its polymerization, modification, and translocation/secretion process.…”

A Polysaccharide Lyase from Stenotrophomonas maltophilia with a Unique, pH-regulated Substrate Specificity

“…For poly-ManA the specific activity (68.5 Ϯ 2.9 units/mg at pH 9) was also significant (Fig. 5B), being within an order of magnitude to the reported values for poly-ManA-specific lyases (31,46,47). Likewise, whereas the specific activity for HA (42.3 Ϯ 1.3 units/mg at pH 5) was the lowest for the three major substrates (Fig.…”

Structure of a PL17 Family Alginate Lyase Demonstrates Functional Similarities among Exotype Depolymerases

“…Determination of the kinetic parameters for degradation of low viscosity alginate by Alg17c was carried out using the thiobarbituric acid assay method ( (23) and Atu3025 from A. tumefaciens (9). Kinetic parameters for wild-type and site-specific active site variants of Alg17c are reported in Table 2 and discussed in the relevant sections below.…”