0 avis
Characterization of substrate and product specificity of the purified recombinant glycogen branching enzyme of Rhodothermus obamensis
Archive ouverte
Background: Glycogen and starch branching enzymes catalyze the formation of alpha(1 -> 6) linkages in storage polysaccharides by rearrangement of preexisting alpha-glucans. This reaction occurs through the cleavage of alpha(1 -> 4) linkage and transfer in alpha(1 -> 6) of the fragment in non-reducing position. These enzymes define major elements that control the structure of both glycogen and starch. Methods: The kinetic parameters of the branching enzyme of Rhodothermus obamensis (RoBE) were established after in vitro incubation with different branched or unbranched alpha-glucans of controlled structure. Results: A minimal chain length of ten glucosyl units was required for the donor substrate to be recognized by RoBE that essentially produces branches of DP 3-8. We show that RoBE preferentially creates new branches by intermolecular mechanism. Branched glucans define better substrates for the enzyme leading to the formation of hyper-branched particles of 30-70 nm in diameter (dextrins). Interestingly, RoBE catalyzes an additional alpha-4-glucanotransferase activity not described so far for a member of the GH13 family. Conclusions: RoBE is able to transfer alpha(1 -> 4)-linked-glucan in C4 position (instead of C6 position for the branching activity) of a glucan to create new alpha(1 -> 4) linkages yielding to the elongation of linear chains subsequently used for further branching. This result is a novel case for the thin border that exists between enzymes of the GH13 family. General significance: This work reveals the original catalytic properties of the thermostable branching enzyme of R. obamensis. It defines new approach to produce highly branched alpha-glucan particles in vitro. (C) 2012 Elsevier B.V. All rights reserved.
Consulter en ligne
Suggestions
Du même auteur
