title: The recombinant Azotobacter vinelandii mannuronan C-5-epimerase AlgE4 epimerizes alginate by a nonrandom attack mechanism.
abstract: The Ca2+-dependent mannuronan C-5-epimerase AlgE4 is a representative of a family of Azotobacter vinelandii enzymes catalyzing the polymer level epimerization of beta-D-mannuronic acid (M) to alpha-L-guluronic acid (G) in the commercially important polysaccharide alginate. The reaction product of recombinantly produced AlgE4 is predominantly characterized by an alternating sequence distribution of the M and G residues (MG blocks). AlgE4 was purified after intracellular overexpression in Escherichia coli, and the activity was shown to be optimal at pH values between 6.5 and 7.0, in the presence of 1-3 mM Ca2+, and at temperatures near 37 degrees C. Sr2+ was found to substitute reasonably well for Ca2+ in activation, whereas Zn2+ strongly inhibited the activity. During epimerization of alginate, the fraction of GMG blocks increased linearly as a function of the total fraction of G residues and comparably much faster than that of MMG blocks. These experimental data could not be accounted for by a random attack mechanism, suggesting that the enzyme either slides along the alginate chain during catalysis or recognizes a pre-existing G residue as a preferred substrate in its consecutive attacks.
authors.affiliation: UNIGEN Center for Molecular Biology, Norwegian University of Science and Technology, N-7489 Trondheim, Norway.
authors.forename: H K
authors.initials: HK
authors.lastname: Høidal
authors.forename: H
authors.initials: H
authors.lastname: Ertesvåg
authors.forename: G
authors.initials: G
authors.lastname: Skjåk-Braek
authors.forename: B T
authors.initials: BT
authors.lastname: Stokke
authors.forename: S
authors.initials: S
authors.lastname: Valla
mesh_terms: D016948:Azotobacter vinelandii / Q000201:enzymology*; D002238:Carbohydrate Epimerases / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D011994:Recombinant Proteins; D002238:Carbohydrate Epimerases; C086519:mannuronan c-5-epimerase