Abstract
Structural and functional analyses of alginate lyases are important in the clarification of the biofilm-dependent ecosystem in Pseudomonas aeruginosa and in the development of therapeutic agents for bacterial disease. Most alginate lyases are classified into polysaccharide lyase (PL) family-5 and -7 based on their primary structures. Family PL-7 enzymes are still poorly characterized especially in structural properties. Among family PL-7, a gene coding for a hypothetical protein (PA1167) homologous to Sphingomonas alginate lyase A1-II was found to be present in the P. aeruginosa genome. PA1167 overexpressed in Escherichia coli cleaved glycosidic bonds in alginate and released unsaturated saccharides, indicating that PA1167 is an alginate lyase catalyzing a beta-elimination reaction. The enzyme acted preferably on heteropolymeric regions endolytically and worked most efficiently at pH 8.5 and 40 degrees C. The specific activity of PA1167, however, was much weaker than that of the known alginate lyase AlgL, suggesting that AlgL plays a main role in alginate depolymerization in P. aeruginosa. In addition to this specific activity, differences were found between PA1167 and AlgL in enzyme properties such as molecular mass, optimum pH, salt effect, and substrate specificity. The first crystal structure of the family PL-7 alginate lyase was determined at 2.0 A resolution. PA1167 was found to form a glove-like beta-sandwich composed of 15 beta-strands and 3 alpha-helices. The structural difference between the beta-sandwich PA1167 of family PL-7 and alpha/alpha-barrel AlgL of family PL-5 may be responsible for the enzyme characteristics. Crystal structures of polysaccharide lyases determined so far indicate that they can be assigned to three folding groups having parallel beta-helix, alpha/alpha-barrel, and alpha/alpha-barrel + antiparallel beta-sheet structures as basic frames. PA1167 is the fourth novel folding structure found among polysaccharide lyases.
Highlights
Structural and functional analyses of alginate lyases are important in the clarification of the biofilm-dependent ecosystem in Pseudomonas aeruginosa and in the development of therapeutic agents for bacterial disease
Among family polysaccharide lyase (PL)-7, a gene coding for a hypothetical protein (PA1167) homologous to Sphingomonas alginate lyase A1-II was found to be present in the P. aeruginosa genome
P. aeruginosa produces the periplasmic alginate lyase AlgL, which is specific for poly(M) [42] and belongs to family PL-5
Summary
Structural and functional analyses of alginate lyases are important in the clarification of the biofilm-dependent ecosystem in Pseudomonas aeruginosa and in the development of therapeutic agents for bacterial disease. The specific activity of PA1167, was much weaker than that of the known alginate lyase AlgL, suggesting that AlgL plays a main role in alginate depolymerization in P. aeruginosa In addition to this specific activity, differences were found between PA1167 and AlgL in enzyme properties such as molecular mass, optimum pH, salt effect, and substrate specificity. Polysaccharide lyases recognize uronic acid residues in polysaccharides and catalyze a -elimination reaction by releasing unsaturated saccharides with CϭC double bonds at nonreducing terminal uronate residues These characteristics of lyases indicate that they share common structural features determining their uronate-recognition sites and reaction modes (-elimination reaction). Based on their primary structures, polysaccharide lyases are classified into 14 families (PL1-1–14), each of which includes various function-unknown proteins that have sequence homology in the Carbohydrate-Active enZYme (CAZY) data base (afmb.cnrs-mrs.fr/ϳcazy/CAZY/index.html). Bacterial alginate plays an important role in the colonization of target cells
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