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.

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Alginate is a linear polysaccharide composed of ␤-D-mannuronate and the C 5 epimer ␣-L-guluronate, arranged in three different ways: poly-␤-D-mannuronate (polyM), poly-␣-L-guluronate (polyG), and heteropolymeric (polyMG) region in which there is a random arrangement of the monomers (4). Alginate produced by brown seaweeds is not acetylated and is widely used in food and pharmaceutical industries because of the ability of the polymer to chelate metal ions and form a highly viscous solution (18). The acetylated form of alginate is synthesized by certain bacteria, such as mucoid cells of Pseudomonas aeruginosa and Azotobacter vinelandii. P. aeruginosa causes serious chronic pulmonary infections in the lungs of patients with cystic fibrosis (CF) (2), and alginate produced by the bacterial cells seems to play a crucial role in the adherence of the bacterium to target cells (21). The alginate functions as a biofilm and decreases the effect of antimicrobial agents by repressing the penetration of the agent into the biofilm, thus making it difficult to treat biofilm-dependent bacterial infectious diseases (5).A bacterium, Sphingomonas sp. strain A1, incorporates alginate into the cells through a pit formed on the cell surface (8), and an ABC (ATP-binding cassette) transporter specific to the macromolecule is responsible for transport of alginate (16) (Fig. 1). The incorporated alginate is depolymerized by three types of cytoplasmic alginate-depolymerizing enzymes (alginate lyases A1-I [66 kDa], A1-II [25 kDa], and A1-III [40 kDa]) formed from a common precursor protein through the consecutive processes of posttranslational modification (Fig. 1) (17). A1-I is autoprocessed to give rise A1-II and A1-III (9), and these alginate lyases cleave glycosidic bonds endolytically in the alginate molecule by ␤-elimination reaction. Briefly, A1-I is active on acetylated and nonacetylated alginates. A1-II prefers polyG and nonacetylated alginate produced by brown seaweeds. A1-III efficiently liquefies polyM and acetylated alginates produced by mucoid cells of P. aeruginosa derived from the lungs of CF patients (17, 32); this property may be useful as a clinical agent for the therapy of CF and other infectious diseases caused by P. aeruginosa.Three alginate lyases (A1-I, A1-II, and A1-III) can produce di-and trisaccharides from alginate as major final products (6, 32), thus implying that the cells of Sphingomonas sp. strain A1 have an additional enzyme responsible for the degradation of alginate oligosaccharides to the constituent monosaccharides. To construct the complete metabolic pathway for alginate in Sphingomonas sp. strain A1, we have purified and characterized the enzyme (oligoalginate lyase) catalyzing the degradation of alginate oligosaccharides and determined its primary structure by cloning the gene. MATERIALS AND METHODSMaterials. Sodium alginate (average molecular weight, 25,700; polymerization degree, more than 100, viscosity, 1,000 cp) from Eisenia bicyclis and DEAEcellulose were purchased from Nacalai Tesque Co. Ltd.,...