Structure-Function Studies of Adpglucose Pyrophosphorylase from Thermodesulfovibrio yellowstonii: Probing the Role of the C-Terminus in Allosteric Regulatio

ADP-glucose pyrophosphorylase (ADPG PPase) functions as the rate-limiting enzyme in the glucan biosynthesis pathways and its successful engineering would increase the yield of biodegradable and renewable carbon. Multiple sequence alignments revealed deviations from consensus in the N-terminal catalytic and C-terminal allosteric region of the unique Thermodesulfovibrio yellowstonii (Td. y) ADPG PPase. The C-terminus region has been shown to be involved in allosteric regulation of plant ADPG PPases but its role in bacterial forms of the enzyme is relatively unexplored. The Td.y charge disrupting mutations, K365G and R397A, were generated by site-directed mutagenesis and purified to homogeneity. The positively charged Lys at 365 and Arg at 397 were hypothesized to contribute to PEP binding and activation. Kinetic studies revealed that the K365G protein displayed a lower Vmax (∼22 % of wild-type [WT]) but ∼2-fold higher apparent affinity for ATP in the absence of effectors. While the wild type enzyme was found to be activated by PEP, G6P, and 3PGA in ATP saturation studies, the K365G protein displayed inhibition by PEP and G6P. In the absence of effectors, the R397A displayed a similar apparent affinity for ATP compared to WT but a 24-fold decrease in Vmax. The major effect of the R397A protein was the conversion of WT activators (3PGA and FBP) to inhibitors. The results indicate that K365 and R397 are critical for PEP activation while also influencing other allosteric properties. Initial crystallization trials for the R397A enzyme resulted in a crystal with 3.0 A.