Influence of expansin carbohydrate binding module on in vitro polygalacturonase enzyme activity

CRISTINA F. NARDI; CRISTIAN M. ESCUDERO; NATALIA M. VILLARREAL; GUSTAVO A. MARTÍNEZ; PEDRO M. CIVELLO

San Miguel de Tucumán - Tucumán - Argentina

Congreso; XLV Reunión Anual - Sociedad Argentina de Investigación en Bioquímica y Biología Molecular; 2009

SAIB (Sociedad Argentina de Investigación en Bioquímica y Biología Molecular)

Fruit softening is associated with cell wall disassembly mediated by the action of a set of enzymes and proteins. Expansins (EXPs), a group of proteins with unknown enzymatic activity, and polygalacturonases (PGs) are proposed to be involved in this process. It has been suggested that EXPs and PGs could cooperatively participate in ripening-associated cell wall degradation, though there is scarce experimental evidence that support such statement. FAEXP2 encodes a putative strawberry expansin (FAEXP2) with a predicted carbohydrate binding module (CBM) in its C-terminal domain and an ?expansin domain? in its N-terminal domain. In order to evaluate the influence of expansin, we performed in vitro PG activity assays in the presence of the full length protein (FAEXP2) or its CBM (CBM-FAEXP2). Both cDNAs were cloned, over-expressed in E. coli, and the recombinant polypeptides were purified. In vitro PG activity was measured using enzymatic extract from tomato, citrus pectin as substrate and the addition of FAEXP2 or CBM-FAEXP2. The presence of CBM-FAEXP2 in the reaction mixture decreased PG activity, and a slighter effect was found in the case of FAEXP2 also. These results suggest that CBM-FAEXP2 could interact with pectins and then interfere with the activity of polygalacturonase enzyme.