Structure‐function studies of ADPGlucose Pyrophosphorylase from Thermodesulfovibrio yellowstonii: Probing the Role of the C‐terminus in Allosteric Regulation

Abstract

Starch serves as both a critical food source and an important biodegradable and renewable carbon source for biofuel and other industries including adhesives, packaging material, and cosmetics. ADP‐glucose pyrophosphorylase (ADPG PPase, glgC gene product) is the key enzyme that is responsible for the rate‐limiting step of both glycogen and starch biosynthesis in bacteria and plants, catalyzing the conversion of glucose‐1‐phosphate and ATP to ADPG and pyrophosphate. Thermodesulfovibrio yellowstonii (Td. y) is a thermophilic sulfate‐reducing bacterium that was isolated from geothermal vents in Yellowstone National Park. Little is known about this ADPG PPase which displays increased heat stability, salt tolerance, and altered regulation compared to other ADPG PPase family members. The Td.y enzyme is substantially activated by phosphoenolpyruvate (PEP), which differs from many other ADPG PPases, as well as G6P, 3PGA, and sulfate. These properties could contribute to biomass production in different crop environments. Multiple sequence alignments have shown deviations from consensus residues in the N‐terminal allosteric region and C‐terminal region of the Td.y ADPG PPase. Two of the more interesting differences are at positions 365 and 397, which have been shown to be involved in allosteric regulation of other ADPG PPases, as part of a conserved region in the enzyme family and associated with plant enzymes, respectively. The K365G and R397A enzymes were generated by site‐directed mutagenesis as a first step in probing structure‐function relationships. We hypothesize that the positively charged lysine at 365 and arginine at 397 may contribute to PEP binding and activation. The altered proteins were purified to homogeneity by a procedure that included two hydroxyapatite chromatography steps followed by size exclusion chromatography. Initial kinetic studies revealed that the K365G protein displayed a lower Vmax (~25% of wild‐type [WT]) but ~2‐fold higher apparent affinity for ATP in the absence of effectors. In contrast to the WT, which was activated over 3‐fold by PEP, the K365G enzyme only exhibited ~10% activation by PEP and a decrease in apparent affinity for ATP. Conversely, the K365G enzyme was activated 2.6 fold by sulfate, an effector which has a much smaller effect on the Vmax of WT. In the absence of effectors, the R397A displayed a similar apparent affinity for ATP compared to WT but a 24 fold decrease in Vmax. In the presence of PEP, R397A exhibited lower apparent affinity for ATP and a Vmax decreased ~42 fold. The response of this enzyme to G6P was very similar to WT. The results so far support the hypothesis that K365 and R397 are critical for PEP activation while also influencing other allosteric properties. Complete kinetic analyses of the K365G and R397A enzymes as well as crystallization trials are underway.Support or Funding InformationSupported in part by NSF and NSF BIO MCB grant #0448676.