Mapping enzymatic functionalities of mannuronan C-5 epimerases and their modular units by dynamic force spectroscopy.
Alginates are (1-->4)-linked structural copolyuronans consisting of beta-D-mannuronic acid (M) and its C-5 epimer alpha-L-guluronic acid (G). The residue sequence variation is introduced in a unique postpolymerisation step catalysed by a family of C-5 epimerases named AlgE enzymes. The seven known AlgE's are composed of two modules, designated A and R, present in different number. The molecular details of the structure-function relationship of these seven epimerases, introducing specific residue sequences, are not understood. In this study, single-molecular pair interactions between alginate and AlgE enzymes were investigated using dynamic force spectroscopy. The AlgE enzymes AlgE4 and AlgE6, the recombinant construct PKA1 composed of A- and R-modules from various AlgE's, as well as separate R- and A-modules were studied. The strength of the protein-mannuronan interaction, when applying a loading rate of 0.6 nN/s, varied from 73 pN (AlgE4) to 144 pN (A-module). The determined potential width, that is, the distance from the activation barrier to the bound substrate molecule, was 0.23 nm for AlgE4, 0.19 nm for AlgE6 and 0.1 nm for the A-module. No attraction was observed between the R-module and the substrate. The observations indicate that the A-module contains the substrate binding site and that the R-module modulates the enzyme-substrate binding strength. The observed AlgE4-polymer residence times, two orders of magnitude longer than expected from kcat reported for AlgE4, not observed for PKA1, led us to propose a processive mode of action of AlgE4.
Sletmoen
Marit
M
Biophysics and Medical Technology, Department of Physics, The Norwegian University of Science and Technology, NTNU, NO-7491 Trondheim, Norway.
Skjåk-Braek
Gudmund
G
Stokke
Bjørn T
BT
D000464:Alginates / Q000737:chemistry; D002236:Carbohydrate Conformation; D002238:Carbohydrate Epimerases / Q000737:chemistry*; D002240:Carbohydrate Sequence; D018625:Microscopy, Atomic Force / Q000379:methods*; D008969:Molecular Sequence Data; D025941:Protein Interaction Mapping / Q000379:methods*
D000464:Alginates; D002238:Carbohydrate Epimerases; C086519:mannuronan c-5-epimerase