Construction, expression and characterization of chimeric ADP-glucose pyrophosphorylases made using the potato tuber and cyanobacterial enzymes

BALLICORA MA, SESMA JJ, STEIN J, GÁRRIZ A, IGLESIAS AA AND PREISS J

San Francisco, California, USA

Congreso; Biochemistry and Molecular Biology- New World Science for the Next Millenium; 1999

Federation of American Societies for Experimental Biology

ADP-glucose pyrophosphorylase (AGPase) from cyanobacteria and from higher plants have similar regulatory properties and homologous sequences. However, the cyanobacterial enzyme is a homotetramer, whereas the plant enzymes are heterotetrameric composed by two different subunits. To study the domains of these proteins we constructed chimeric enzymes: PSSAna contains 292 amino acids from the N-terminal of the small subunit of AGPase from potato tuber (PSS) and 159 amino acids from the C-terminal of the enzyme from Anabaena PCC 7120. The first region contains the amino acids that have been identified as part of the catalytic site and the second, residues that are located on the regulatory site. AnaPSS is the inverted construction. Plasmids containing these chimeric genes were expressed alone or together with the compatible plasmid that contains the gene for the large subunit of AGPase from potato tuber (PLS). These chimeric proteins were purified and characterized. Results suggest there is a great structural homology between the enzyme from Anabaena and PSS since one regulatory domain can work with the catalytic domain of the other. However, PSSAna and ANAPSS have intermediate kinetic properties rather than the regulatory characteristics from one enzyme and the catalytic from the other suggesting that the regulation also depends on the interaction between the N- and C- terminal.