Thermus thermophilus glycoside hydrolase family 57 branching enzyme: Crystal structure, mechanism of action, and products formed

Marta Palomo, Tjaard Pijning, Thijs Booiman, Justyna M. Dobruchowska, Jeroen Van Der Vlist, Slavko Kralj, Antoni Planas, Katja Loos, Johannis P. Kamerling, Bauke W. Dijkstra, Marc J.E.C. Van Der Maarel, Lubbert Dijkhuizen, Hans Leemhuis

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Resum

Branching enzyme (EC 2.4.1.18; glycogen branching enzyme; GBE) catalyzes the formation of α1,6-branching points in glycogen. Until recently it was believed that all GBEs belong to glycoside hydrolase family 13 (GH13). Here we describe the cloning and expression of the Thermus thermophilus family GH57-type GBE and report its biochemical properties and crystal structure at 1.35-Å resolution. The enzyme has a central (β/α)7-fold catalytic domain A with an inserted domain B between β2 and α5 and an α-helix-rich C-terminal domain, which is shown to be essential for substrate binding and catalysis. A maltotriose was modeled in the active site of the enzyme which suggests that there is insufficient space for simultaneously binding of donor and acceptor substrates, and that the donor substrate must be cleaved before acceptor substrate can bind. The biochemical assessment showed that the GH57 GBE possesses about 4% hydrolytic activity with amylose and in vitro forms a glucan product with a novel fine structure, demonstrating that the GH57 GBE is clearly different from the GH13 GBEs characterized to date.

Idioma originalAnglès
Pàgines (de-a)3520-3530
Nombre de pàgines11
RevistaJournal of Biological Chemistry
Volum286
Número5
DOIs
Estat de la publicacióPublicada - 4 de febr. 2011

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