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 beta-d-mannuronic acid and its C-5 epimer alpha-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-A resolution. AlgE4A folds into a right-handed parallel beta-helix structure originally found in pectate lyase C and subsequently in several polysaccharide lyases and hydrolases. The beta-helix is composed of four parallel beta-sheets, comprising 12 complete turns, and has an amphipathic alpha-helix near the N terminus. The catalytic site is positioned in a positively charged cleft formed by loops extending from the surface encompassing Asp(152), an amino acid previously shown to be important for the reaction. Site-directed mutagenesis further implicates Tyr(149), His(154), and Asp(178) as being essential for activity. Tyr(149) probably acts as the proton acceptor, whereas His(154) is the proton donor in the epimerization reaction.
Laboratory of Biophysical Chemistry, GBB, University of Groningen, Nijenborgh 4, Groningen, The Netherlands.
Henriëtte J
HJ
Rozeboom
Tonje M
TM
Bjerkan
Kor H
KH
Kalk
Helga
H
Ertesvåg
Synnøve
S
Holtan
Finn L
FL
Aachmann
Svein
S
Valla
Bauke W
BW
Dijkstra
D016948:Azotobacter vinelandii / Q000201:enzymology*; D002238:Carbohydrate Epimerases / Q000737:chemistry*; D020134:Catalytic Domain / Q000502:physiology*; D018360:Crystallography, X-Ray / Q000379:methods; D017433:Protein Structure, Secondary / Q000502:physiology; D040681:Structural Homology, Protein
D002238:Carbohydrate Epimerases; C086519:mannuronan c-5-epimerase