Rapid purification of the potato ADP-glucose pyrophosphorylase by polyhistidine-mediated chromatography.

In an attempt to obtain facile methods to purify the heterotetrameric ADP-glucose pyrophosphorylase (AGPase), polyhistidine tags were attached to either the large (LS) or small (SS) subunits of this oligomeric enzyme. The addition of polyhistidine tag to the N-terminus of the LS or SS and co-expression with its unmodified counterpart subunit resulted in substantial induction of enzyme activity. In contrast, attachment of a polyhistidine-containing peptide through the use of a commercially available pET vector or addition of polyhistidine tags to the C-terminal ends of either subunit resulted in poor expression and/or production of enzyme activity. Preliminary experiment showed that these polyhistidine N-terminal-tagged enzymes interacted with Ni-NTA-agarose, indicating that immobilized metal affinity chromatography (IMAC) would be useful for efficient purification of the heterotetrameric AGPases. When ion-exchange chromatography step was employed prior to the IMAC, the polyhistidine-tagged AGPases were purified to near homogeneity. Comparison of kinetic parameters between AGPases with and without the polyhistidine tags revealed that attachment of the polyhistidine did not alter the allosteric and catalytic properties of the enzymes. These results indicate that polyhistidine tags will be useful for the rapid purification of preparative amounts of AGPases for biochemical and physical studies.

Directed molecular evolution of ADP-glucose pyrophosphorylase.

ADP-glucose pyrophosphorylase catalyzes a rate-limiting reaction in prokaryotic glycogen and plant starch biosynthesis. Despite sharing similar molecular size and catalytic and allosteric regulatory properties, the prokaryotic and higher plant enzymes differ in higher-order protein structure. The bacterial enzyme is encoded by a single gene whose product of ca. 50,000 Da assembles into a homotetrameric structure. Although the higher plant enzyme has a similar molecular size, it is made up of a pair of large subunits and a pair of small subunits, encoded by different genes. To identify the basis for the evolution of AGPase function and quaternary structure, a potato small subunit homotetrameric mutant, TG-15, was subjected to iterations of DNA shuffling and screened for enzyme variants with up-regulated catalytic and/or regulatory properties. A glycogen selection/screening regimen of buoyant density gradient centrifugation and iodine vapor colony staining on glucose-containing media was used to increase the stringency of selection. This approach led to the isolation of a population of AGPase small subunit homotetramer enzymes with enhanced affinity toward ATP and increased sensitivity to activator and/or greater resistance to inhibition than TG-15. Several enzymes displayed a shift in effector preference from 3-phosphoglycerate to fructose-6 phosphate or fructose-1,6-bis-phosphate, effectors used by specific bacterial AGPases. Our results suggest that evolution of AGPase, with regard to quaternary structure, allosteric effector selectivity, and effector sensitivity, can occur through the introduction of a few point mutations alone with low-level recombination hastening the process.