Mode of action of recombinant Azotobacter vinelandii mannuronan C-5 epimerases AlgE2 and AlgE4

Abstract

The enzymes mannuronan C-5 epimerases catalyze conversion of beta-D-mannuronic acid to alpha-L-guluronic acid in alginates at the polymer level and thereby introduce sequences that have functional properties relevant to gelation. The enzymatic conversion by recombinant mannuronan C-5 epimerases AlgE4 and AlgE2 on alginate type substrates with different degree of polymerization and initial low fraction of alpha-L-guluronic acid was investigated. Essentially no enzymatic activity was found for fractionated mannuronan oligomer substrates with an average degree of polymerization, DP(n), less than or equal 6, whereas increasing the DP(n) yielded increased epimerization activity. This indicates that these enzymes have an active site consisting of binding domains for consecutive residues that requires interaction with 7 or more consecutive residues to show enzymatic activity. The experimentally determined kinetics of the reaction, and the residue sequence arrangement introduced by the epimerization, were modeled using Monte Carlo simulation accounting for the various competing intrachain substrates and assuming either a processive mode of action or preferred attack. The comparison between experimental data and simulation results suggests that epimerization by AlgE4 is best described by a processive mode of action, whereas the mode of action of AlgE2 appears to be more difficult to determine.

Single molecule investigation of the onset and minimum size of the calcium-mediated junction zone in alginate

“…Since M and G are epimers of each other, the alginate polymer is synthesised as polymannuronic acid and epimerisation of individual residues within the polymannuronic acid chain to guluronic acids is carried out by a family of epimerases (Haug & Larsen, 1971a, b;. This enzymatic epimerisation results in varied patterns of the sequences GMG and GG being generated within the polymannuronic acid, with different organisms having different epimerases that make alginates specific to each species Hartmann, Holm, Johansen, Skjåk-Braek, & Stokke, 2002;Høidal, Ertesvåg, Skjåk-Braek, Stokke, & Valla, 1999;Holtan, Zhang, Strand, & Skjåk-Braek, 2006). …”

Structural and Functional Characterization of the R-modules in Alginate C-5 Epimerases AlgE4 and AlgE6 from Azotobacter vinelandii

“…R-modules alone show no catalytic activity on alginates; nevertheless, if one R-module is bound after an A-module, the epimerization rate is increased 10-fold (16). Epimerization of alginate by AlgE4 occurs by a processive mode of action meaning that the enzyme is sliding along the alginate polymer and where both the A-module and the R-module bind to the alginate molecule (17)(18)(19). Therefore, it was suggested that the R-module helps to orient the alginate in the active site and to keep AlgE4 tightly bound to the polymer during epimerization.…”

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

“…For longer chains the reaction proceeds toward the reducing end in a non-random, processive manner (20). On average, the enzyme epimerizes 10 units ((MG) 10 ) in each reaction before leaving the chain (19).…”