Structural characterization of the three ATXYN1 CBMs from Arabidopsis thaliana

GRISOLIA, M.J.; BUSTI, P.; BARCHIESI, J.; GOMEZ-CASATI, D.F; DELORENZI, N; BUSI, M.V.

CABA, Buenos Aires

Congreso; Reunión Satélite de la Sociedad Argentina de Investigación en Bioquímica y Biología Molecular (SAIB); 2013

SAIB

The Arabidopsis thaliana genome codifies for the xylanase (AtXyn1), which is predominantly expressed in vascular bundles, but not in vessel cells. Like many polysaccharide-degrading enzymes, AtXyn1 displays a modular structure in which the catalytic domain (C-terminal) is attached to three in tandem non-catalytic modules. Those modules where classified in the family 22 (previously family 4_9) of carbohydrate binding modules (CBM) (http://www.cazy.org/CBM22.html), according to sequence similarity. In order to determine if those domains were classified correctly, we performed a sequence and structural analysis, leading to an acceptabletridimensional model of AtXyn1 consisting in a beta-sandwich (classic beta -jelly roll) with a shallow surface groove that forms the ligandbinding site. Those models were conducted employing a ?threading? method using the two CBMs form the Clostridium thermocellum (family 22) xylanase (XylY) (UniProt=XYNY_CLOTM) as template (showing a 17% of identity with the module Xyl2 fromAtXyn1). Then, we cloned the three CMBs in the pET28 vector and the protein was expressed, purified and the physicochemical and structural parameters in the absence or presence of severalligands were determined. We conclude that the CMBs of AtXyn1 are correctly classified since its folding and affinity to xylane are consistent with the rest of the members of the CAZy family 22.