The recombinant Azotobacter vinelandii mannuronan C-5-epimerase AlgE4 epimerizes alginate by a nonrandom attack mechanism

The Ca2+-dependent mannuronan C-5-epimerase AlgE4 is a representative of a family of Azotobacter vinelandii enzymes catalyzing the polymer level epime rization of beta-D-mannuronic acid (M) to alpha-L-guluronic acid (G) in the commercially important polysaccharide alginate. The reaction product of re combinantly produced AlgE4 is predominantly characterized by an alternating sequence distribution of the M and Gr residues (MG blocks). AlgE4 was puri fied after intracellular overexpression in Escherichia coli, and the activi ty was shown to be optimal at pH values between 6.5 and 7.0, in the presenc e 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 in hibited the activity. During epimerization of alginate, the fraction of GMG blocks increased linearly as a function of the total fraction of Gr residu es and comparably much faster than that of MMG blocks. These experimental d ata could not be accounted for by a random attack mechanism, suggesting tha t the enzyme either slides along the alginate chain during catalysis or rec ognizes a pre-existing G; residue as a preferred substrate in its consecuti ve attacks.