Dang-Quan Zhang, Jin-Fa Wang, Xiao-Feng Tan
Although carrot antifreeze protein (DcAFP) has a strong antifreeze activity, it also belongs to the plant polygalacturonase (PGase)-inhibiting protein (PGIP) family based on sequence similarities, including the presence of a typical leucine-rich repeat (LRR) motif. In order to determine the mechanistic/structural relationship of DcAFP and PGIP, we used yeast two-hybrid technology to analyze whether carrot AFP could act as a PGIP. The complete DcAFP and PGase encoding sequences obtained from fungus Alternaria alternate by RT-PCR were cloned into the bait and capture vectors, respectively, and yeast two-hybrid assays were performed. The results revealed that there was no evidence for an interaction between DcAFP and PGase, which suggests that DcAFP likely lacks PGIP activity. An analysis of the electrostatic potential of DcAFP and other PGIPs revealed that many non-conservative acidic residues within the β-helix of the DcAFP LRR motif had been substituted to basic amino acids, changing the surface from negative to positive. This will prevent electrostatic binding of DcAFP with the positively charged activity surface of PGase. It is confirmed that nonconservative amino acids within the LRR motif of the carrot antifreeze protein are correlated with its activity loss of inhibiting polygalacturonase.
Dang-Quan Zhang, , Jin-Fa Wang, and Xiao-Feng Tan, (2007). CARROT ANTIFREEZE PROTEIN CANNOT ACT AS A PLANT PGIP. Acta Hortic. 763, 267-274
DOI: 10.17660/ActaHortic.2007.763.35
polygalacturonase-inhibiting protein, leucine-rich repeat, yeast two-hybrid

Acta Horticulturae