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.

Figure 1

Activity of AlgL as a function of protein stock concentration. The polyM concentration was 20 μM, and had an average dp of 34. Each reaction was initiated by the addition of 30 μg of AlgL from a stock solution at the concentration indicated.

Figure 2

Steady state kinetic parameters for AlgL-catalyzed degradation of polyM as a function of substrate length. A) k_cat; B) K_m; C) k_cat/K_m.

Figure 2

Steady state kinetic parameters for AlgL-catalyzed degradation of polyM as a function of substrate length. A) k_cat; B) K_m; C) k_cat/K_m.

Figure 2

Steady state kinetic parameters for AlgL-catalyzed degradation of polyM as a function of substrate length. A) k_cat; B) K_m; C) k_cat/K_m.

Figure 3

¹H NMR spectra of PDO486 alginate before (A) and after (B) digestion with AlgL. G and M refer to guluronate and mannuronate, respectively; the number is the proton from which the signal arises. Δ-G and Δ-M refer to unsaturated residues at the nonreducing end of the products adjacent to guluronate and mannuronate, respectively. The area under each labeled peak is given below the chemical shift axis.

Figure 4

Pre-steady state product formation by AlgL. Products were separated from the quenched reaction by HPLC and quantitated as described in the text.

Scheme 1

Scheme 2