Expression, Purification, and Initial Characterization of the Diverse ADPGlucose Pyrophosphorylase from Thermodesulfovibrio yellowstonii

Abstract

ADP‐Glucose pyrophosphorylase (ADPG PPase, glgC gene product) catalyzes the rate‐limiting step of glucan biosynthesis in plants and bacteria. Structure‐function studies of ADPG PPases from diverse organisms will allow for the identification of the amino acids responsible for different characteristics and the subsequent engineering of a highly active form of the enzyme to increase the supply of renewable carbon. The glgC gene from Thermodesulfovibrio yellowstonii (Td.y.) was successfully cloned and expressed. This enzyme displays only ~30% identity to the well characterized E. coli and A. tumefaciens (Ag.t.) enzymes, and harbors unusual sequences compared to other ADPG PPases in regions known to be important for allosteric regulation. Interestingly, molecular modeling predicted that the structure was more similar to the potato tuber enzyme (PDB 1yp3) than the bacterial Ag.t. ADPG PPase structure (PDB 3brk). The enzyme was successfully purified by sonication, a heat step, anion exchange, and hydrophobic interaction chromatography. The Td. y. enzyme was found to be stable up to 75 °C, and exhibited optimal activity at pH 8.5 and 70 °C. The enzyme was also activated by 2 mM G6P (~5‐fold), FBP (~3‐fold), sulfate (~4‐fold) and strongly inhibited (80%) by 2 mM AMP, indicating a novel allosteric specificity pattern. Supported by NSF Award 0448676.