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Mode of action of recombinant Azotobacter vinelandii mannuronan C‐5 epimerases AlgE2 and AlgE4
Author(s) -
Hartmann Martin,
Holm Olav B.,
Johansen Gunn A. B.,
SkjåkBræk Gudmund,
Stokke Bjørn T.
Publication year - 2002
Publication title -
biopolymers
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.556
H-Index - 125
eISSN - 1097-0282
pISSN - 0006-3525
DOI - 10.1002/bip.10017
Subject(s) - epimer , azotobacter vinelandii , chemistry , enzyme , polymerization , residue (chemistry) , recombinant dna , stereochemistry , polysaccharide , degree of polymerization , mode of action , polymer , biochemistry , organic chemistry , gene , nitrogenase , nitrogen fixation , nitrogen
The enzymes mannuronan C‐5 epimerases catalyze conversion of β‐ D ‐mannuronic acid to α‐ 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 α‐ 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. © 2002 John Wiley & Sons, Inc. Biopolymers 63: 77–88, 2002