Single-molecular pair unbinding studies of Mannuronan C-5 epimerase AlgE4 and its polymer substrate.
Alginate biosynthesis involves C-5-mannuronan epimerases catalyzing the conversion of beta-D-mannuronic acid to alpha-L-guluronic acid at the polymer level. Mannuronan epimerases are modular enzymes where the various modules yield specific sequential patterns of the converted residues in their polymer products. Here, the interaction between the AlgE4 epimerase and mannuronan is determined by dynamic force spectroscopy. The specific unbinding between molecular pairs of mannuronan and AlgE4 as well as its two modules, A and R, respectively, was studied as a function of force loading rate. The mean protein-mannuronan unbinding forces were determined to be in the range 73-144 pN, depending on the protein, at a loading rate of 0.6 nN/s, and increased with increasing loading rate. The position of the activation barrier was determined to be 0.23 +/- 0.04 nm for the AlgE4 and 0.10 +/- 0.02 nm for its A-module. The lack of interaction observed between the R-module and mannuronan suggest that the A-module contains the binding site for the polymer substrate. The ratio between the epimerase-mannuronan dissociation rate and the catalytic rate for epimerization of single hexose residues suggests a processive mode of action of the AlgE4 epimerase yielding the observed sequence pattern in the uronan associated with the A-module of this enzyme.
Sletmoen
Marit
M
Section of Biophysics and Medical Technology, Department of Physics, The Norwegian University of Science and Technology, NO-7491 Trondheim, Norway.
Skjåk-Braek
Gudmund
G
Stokke
Bjørn T
BT
D002238:Carbohydrate Epimerases / Q000032:analysis* / Q000737:chemistry*; D002384:Catalysis; D004800:Enzymes, Immobilized / Q000737:chemistry; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D046911:Macromolecular Substances / Q000032:analysis / Q000737:chemistry; D008351:Mannans / Q000032:analysis / Q000737:chemistry; D018625:Microscopy, Atomic Force / Q000379:methods; D011108:Polymers / Q000032:analysis* / Q000737:chemistry*; D011134:Polysaccharides / Q000737:chemistry; D011549:Pseudomonas / Q000201:enzymology; D011994:Recombinant Proteins / Q000032:analysis / Q000737:chemistry; D013379:Substrate Specificity
D004800:Enzymes, Immobilized; D046911:Macromolecular Substances; D008351:Mannans; D011108:Polymers; D011134:Polysaccharides; D011994:Recombinant Proteins; D002238:Carbohydrate Epimerases; C086519:mannuronan c-5-epimerase