Probing the Roles of Phenylalanine‐23 and Glutamate‐15 in the ADP‐Glucose Pyrophosphorylase from Thermodesulfovibrio yellowstonii

ADP‐Glucose Pyrophosphorylase (ADPG PPase) catalyzes the rate limiting step of starch and glycogen biosynthesis in plants and bacteria, respectively. Understanding the structure‐function relationships of ADPG PPase can facilitate rational engineering of this enzyme to increase glucan production. The ADPG PPase from Thermodesulfovibrio yellowstonii (Td.y.) represents a diverse form of the enzyme with some novel properties that could be incorporated into a highly active and stable form of the enzyme. Further, sequence alignment data indicate that Td.y harbors varied amino acids in two conserved N‐terminal region (glycine rich region and RRAKPAV region) that are known to be important for function and regulation in other characterized ADPG PPases. The variants F23R and E15A were generated by site‐directed mutagenesis and successfully expressed and purified using hydroxyapatite and size exclusion chromatography. In the absence of effector molecules, the F23R enzyme displayed a 4 fold decrease in activity and a ~2‐fold increase in the S 0.5 for ATP compared to wild‐type (WT). However, in the presence of the activators PEP and FBP, the ATP apparent affinity was comparable to the WT value; further, there was a ~7 fold increase in fold activation compared to WT. Further, effector screening revealed additional metabolites that activate the altered enzyme including NADP + , F6P, pyruvate, and NADH. Preliminary data suggests that phenylalanine may play a role in enzyme conformation, regulation, and allosteric specificity. Initial characterization is underway for the E15A enzyme and various C‐terminus variants. Supported in part by NSF (BIO MCB 0448676).