Molecular evolution of a homotetrameric enzyme of plant origin from the Solanum tuberosum L. ADP -glucose pyrophosphorylase small subunit

ADP-glucose pyrophosphorylase (AGPase) is the allosterically regulated gateway for carbon entry into transient and storage starch in plants as well as glycogen in bacteria. This enzyme plays an important role in the modulation of photosynthetic efficiency in source tissues and directly determines the level of storage starch in sink tissues, thus influencing overall crop yield potential. 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 by iodine vapor staining. This search strategy led to the isolation of small subunit mutants (SUP-1, TG-15) that had pyrophosphorylase activity in the absence of the large subunit. TG-15 was partially purified and kinetic analysis revealed substrate and effector affinities equal to wild type (WT) heterotetrameric enzyme with the exception of ATP binding. TG-15 was subjected to error-prone PCR and three iterations of DNA shuffling. The goal of this work was to identify amino acid residues that mediate effector binding and also to generate single gene constructs that can be utilized in metabolic engineering attempts to determine the role of AGPase in regulating carbon flux in both source and sink tissue. A selection/screening regimen of buoyant density gradient centrifugation and iodine vapor colony staining on medium containing decreasing concentrations of glucose was utilized to increase the stringency of selection after subsequent iterations of DNA shuffling. Individual mutants exhibited enhanced sensitivity to activator (Km of 12muM) and greater resistance to inhibition (3-PGA Ka/Pi Ki ratio of .001) as well as showing a shift in effector preference (fructose-6 phosphate, fructose-1,6 bis-phosphate). Our results demonstrate that alterations in allosteric effector selectivity as well as sensitivity can be created by directed evolution based solely on point mutations, thereby enabling a large area of novel applications for metabolic engineering.