Structural and Mutational Characterization of the Catalytic A-module of the Mannuronan C-5-epimerase AlgE4 from Azotobacter vinelandii.

Alginate is a family of linear copolymers of (1→4)-linked β-D-mannuronic acid and its C-5 epimer α-L-guluronic acid. The polymer is first produced as polymannuronic acid and the guluronic acid residues are then introduced at the polymer level by mannuronan C-5-epimerases. The structure of the catalytic A-module of the Azotobacter vinelandii mannuronan C-5-epimerase AlgE4 has been determined by x-ray crystallography at 2.1-Å resolution. AIgE4A folds into a right-handed parallel β-helix structure originally found in pectate lyase C and subsequently in several polysaccharide lyases and hydrolases. The β-helix is composed of four parallel β-sheets, comprising 12 complete turns, and has an amphipathic α-helix near the N terminus. The catalytic site is positioned in a positively charged cleft formed by loops extending from the surface encompassing Asp152, an amino acid previously shown to be important for the reaction. Site-directed mutagenesis further implicates Tyr149, His154, and Asp178 as being essential for activity. Tyr149 probably acts as the proton acceptor, whereas His154 is the proton donor in the epimerization reaction. [ABSTRACT FROM AUTHOR]