Isolation and characterization of a higher plant ADP-glucose pyrophosphorylase small subunit homotetramer

ADP-glucose pyrophosphorylase (AGPase) is the allosterically regulated gate way for carbon entry into transient and storage starch in plants as well as glycogen in bacteria. This enzyme plays a key role in the modulation of ph otosynthetic efficiency in source tissues and directly determines the level of storage starch in sink tissues, thus influencing overall crop yield pot ential. AGPase is a tetrameric enzyme; in higher plants it consists of two regulatory large subunits (LS) and two catalytic small subunits (SS), while in cyanobacteria and prokaryotes the enzyme is homotetrameric, The potato SS gene in pML10 was mutated by hydroxylamine and mutants were screened for elevated homotetrameric activity hy iodine vapor staining. This search str ategy led to the isolation of SS mutants (SUP-I, TG-15) that had pyrophosph orylase activity in the absence of the LS, TG-15 has a leucine to phenylala nine change at position 48 (L48F) that corresponds to a phenylalanine resid ue at the analogous position in the Escherichia coli homotetrameric AGPase as well as a valine to isoleucine change at position 59 (V59I), TG-15 was p artially purified and kinetic analysis revealed substrate and effector affi nities equal to wild type heterotetrameric enzyme with the exception of ATP binding. (C) 2000 Federation of European Biochemical Societies, Published by Elsevier Science B.V. All rights reserved.