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J. Biol. Chem., Vol. 279, Issue 30, 31863-31872, July 23, 2004

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Structure and Function of a Hypothetical Pseudomonas aeruginosa Protein PA1167 Classified into Family PL-7

A NOVEL ALGINATE LYASE WITH A -SANDWICH FOLD^*

Masayuki Yamasaki, Satoko Moriwaki, Osamu Miyake, Wataru Hashimoto, Kousaku Murata¶||, and Bunzo Mikami¶

From the Division of Agronomy and Horticultural Science, Graduate School of Agriculture, and Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Uji, Kyoto 611-0011, Japan

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. PA1167 is the fourth novel folding structure found among polysaccharide lyases.

Received for publication, March 4, 2004 , and in revised form, April 28, 2004.

The atomic coordinates and structure factors (code 1VAV) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^* This work was supported in part by grants-in-aid from the Ministry of Education, Science, Sports, and Culture of Japan and by the Program for Promotion of Basic Research Activities for Innovative Biosciences (PROBRAIN) of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ Both authors contributed equally to this work.

^|| To whom correspondence should be addressed. Tel.: 81-774-38-3766; Fax: 81-774-38-3767; E-mail: kmurata{at}kais.kyoto-u.ac.jp.

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