Structure‐Function Studies of the ADPGlucose Pyrophosphorylase from Thermodesulfovibrio yellowstonii

• DOI: 10.1096/fasebj.27.1_supplement.1004.6
• Publication Date: 2021-06-07T12:15:07Z
• Authors: Michael Susoeff; Eric Yik; Sandeep Kaur; Guatam Dua; Matthew Badal; Andrew Orry; Christopher R Meyer
• Keywords: 0301 basic medicine; 03 medical and health sciences

ADPGlucose pyrophosphorylase (ADPG PPase, glgC gene product) catalyzes the rate‐limiting step of glucan biosynthesis in plants and bacteria. Engineering of this enzyme family will allow for the increased production of renewable carbon. The Thermodesulfovibrio yellostonii (Td.y) glgC gene has been successfully cloned and the recombinant enzyme purified. This enzyme displays only ~30% identity to other characterized ADPG PPases and harbors unusual sequences in regions involved in regulation. Molecular modeling studies revealed that the Td.y enzyme was most similar to a plant enzyme, in accord with 3‐phosphoglycerate (3‐PGA) activation. Heat stability up to 75°C required the presence of ATP. Initial kinetic studies were performed at 37°C (pH 7.5) and revealed S0.5 values for ATP and Mg of 8.0 mM and 13.9 mM, respectively, and a Vmax value of 5.33 Units/mg. The metabolites PEP (2mM), glucose‐6‐phosphate ( 2 mM), and 3‐PGA (2 mM), were found to increase the apparent affinity for ATP by 4.2, 10.3, and 3.8‐fold, respectively. PEP also increased the Vmax by 2.0‐fold. Based on alignment studies and molecular modeling, the following site‐directed mutants have been generated to probe regulatory properties: E15S, F18K, F23R, S25A, and S28A. Complete characterization of the native and altered proteins is underway. Supported by NSF Award 0448676 and a CSUPERB Presidents’ Commission Scholars grant to E. Yik and C. R. Meyer.