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 ␤-elimination reaction. The enzyme acted preferably on heteropolymeric regions endolytically and worked most efficiently at pH 8.5 and 40°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 Å resolution. PA1167 was found to form a glove-like ␤-sandwich composed of 15 ␤-strands and 3 ␣-helices. The structural difference between the ␤-sandwich PA1167 of family PL-7 and ␣/␣-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 ␤-helix, ␣/␣-barrel, and ␣/␣-barrel ؉ antiparallel ␤-sheet structures as basic frames.

Alginate lyases depolymerize alginate, a heteropolysaccharide consisting of -l-guluronate and -d-mannuronate, through a -elimination reaction. The alginate lyases A1-II (25 kDa) and A1-II H (25 kDa) from Sphingomonas sp. A1, which belong to polysaccharide lyase family PL-7, exhibit 68% homology in primary structure but have different substrate speci®cities. To determine clearly the structural basis for substrate recognition in the depolymerization mechanism by alginate lyases, both proteins were crystallized at 293 K using the vapourdiffusion method. A crystal of A1-II belonged to space group P2 1 and diffracted to 2.2 A Ê resolution, with unit-cell parameters a = 51.3, b = 30.1, c = 101.6 A Ê , = 100.2 , while a crystal of A1-II H belonged to space group P2 1 2 1 2 1 and diffracted to 1.0 A Ê resolution, with unit-cell parameters a = 34.6, b = 68.5, c = 80.3 A Ê .

Alginate lyase depolymerizes alginate, a heteropolysaccharide consisting of alpha-L-guluronate and beta-D-mannuronate, through a beta-elimination reaction. A protein PA1167 with a molecular mass of 25 kDa produced by Pseudomonas aeruginosa is an alginate lyase classified into polysaccharide lyase family PL-7. The enzyme was crystallized at 293 K in a drop solution comprising 1.4 M sodium chloride, 0.1 M potassium sodium phosphate and 0.1 M 2-morpholinoethanesulfonate-sodium hydroxide pH 6.5 by means of the vapor-diffusion method. The crystals were monoclinic and belonged to space group P2(1), with unit-cell parameters a = 43.4, b = 70.3, c = 67.4 A, beta = 94.5 degrees. Diffraction data were collected to 2.0 A from a single crystal.

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