Subtle structural differences crucial for function in similarly engineered ADP-glucose pyrophosphorylase larger subunit in rice and maize

Abstract

ADP – glucose pyrophosphorylase (AGPase) is a key enzyme for starch synthesis in plants. Heterotetrameric plant AGPase is encoded by two genes, Shrunken-2 (Sh2) and Brittle-2 (Bt2). The Sh2 gene encodes regulatory larger subunit and the Bt2 gene encodes smaller subunit having catalytic properties. A specific mutation in Sh2 gene involving insertion of six nucleotides, without changing the reading frame, resulted in the insertion of two additional amino acid residues serine and tyrosine at specific position at carboxyl end and also in an increase in seed weight up to 11–17%. No increase in seed weight with the same insertion in larger subunit of AGPase enzyme in rice was observed even though the rice and maize subunits have 93% of sequence similarity. In this study, the predicted 3D-structures of larger subunit of normal as well as mutated AGPase in maize and rice, were analyzed and superposed. The segment of six amino acid residues long secondary structure just before the site of insertion of additional amino acids (serine and tyrosine) got reduced in case of mutated maize but not in mutated rice. Therefore, the six residue sequence and corresponding subtle secondary structural difference might be the key factors for functional disparity of engineered larger subunits in the two crops.