Substrate recognition by family 7 alginate lyase from Sphingomonas sp. A1.
Sphingomonas sp. A1 alginate lyase A1-II', a member of polysaccharide lyase family 7, shows a broad substrate specificity acting on poly alpha-L-guluronate (poly(G)), poly beta-D-mannuronate (poly(M)) and the heteropolymer (poly(MG)) in alginate molecules. A1-II' with a glove-like beta-sandwich as a basic scaffold forms a cleft covered with two lid loops (L1 and L2). Here, we demonstrate the loop flexibility for substrate binding and structural determinants for broad substrate recognition and catalytic reaction. The two loops associate mutually over the cleft through the formation of a hydrogen bond between their edges (Asn141 and Asn199). A double mutant, A1-II' N141C/N199C, has a disulfide bond between Cys141 and Cys199, and shows little enzyme activity. Adding dithiothreitol to the enzyme reaction mixture leads to a tenfold increase in its molecular activity, suggesting the significance of flexibility in lid loops for accommodating the substrate into the active cleft. In alginate trisaccharide (GGG or MMG)-bound A1-II' Y284F, the enzyme interacts appropriately with substrate hydroxyl groups at subsites +1 and +2 and accommodates G or M, while substrate carboxyl groups are strictly recognized by specific residues. This mechanism for substrate recognition enables A1-II' to show the broad substrate specificity. The structure of A1-II' H191N/Y284F complexed with a tetrasaccharide bound at subsites -1 to +3 suggests that Gln189 functions as a neutralizer for the substrate carboxyl group, His191 as a general base, and Tyr284 as a general acid. This is, to our knowledge, the first report on the structure and function relationship in family 7.
Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Uji, Kyoto 611-0011, Japan.
Kohei
K
Ogura
Masayuki
M
Yamasaki
Bunzo
B
Mikami
Wataru
W
Hashimoto
Kousaku
K
Murata
D000464:Alginates / Q000737:chemistry / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D018360:Crystallography, X-Ray; D020723:Glucuronic Acid / Q000737:chemistry / Q000378:metabolism; D006603:Hexuronic Acids / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D011133:Polysaccharide-Lyases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D020578:Sphingomonas / Q000201:enzymology*; D013379:Substrate Specificity
D000464:Alginates; D001426:Bacterial Proteins; D006603:Hexuronic Acids; D020723:Glucuronic Acid; D011133:Polysaccharide-Lyases; C016158:poly(beta-D-mannuronate) lyase