Mode of action of recombinant Azotobacter vinelandii mannuronan C-5 epimerases AlgE2 and AlgE4.
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.
Hartmann
Martin
M
Norwegian Biopolymer Laboratory, Department of Biotechnology, The Norwegian University of Science and Technology, NTNU, NO-7491 Trondheim, Norway.
Holm
Olav B
OB
Johansen
Gunn A B
GA
Skjåk-Braek
Gudmund
G
Stokke
Bjørn T
BT
D000464:Alginates / Q000737:chemistry / Q000378:metabolism*; D000595:Amino Acid Sequence; D016948:Azotobacter vinelandii / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D002238:Carbohydrate Epimerases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002240:Carbohydrate Sequence; D002384:Catalysis; D020134:Catalytic Domain; D003198:Computer Simulation; D006603:Hexuronic Acids / Q000737:chemistry; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008433:Mathematics; D008956:Models, Chemical; D015233:Models, Statistical; D008969:Molecular Sequence Data; D009010:Monte Carlo Method; D011994:Recombinant Proteins / Q000378:metabolism*; D013379:Substrate Specificity
D000464:Alginates; D006603:Hexuronic Acids; D011994:Recombinant Proteins; C007896:guluronic acid; D002238:Carbohydrate Epimerases; C086519:mannuronan c-5-epimerase