Probing the role of S28 in Thermodesulfovibrio yellowstonii adenonsine diphosphate glucose pyrophosphorylase: insight in to allosteric regulation and sensitivity (769.14)

Renewable and biodegradable glucans are in demand for use in a variety of industries. Adenosine Diphosphate Glucose Pyrophosphorylase (ADPG PPase), an allosterically regulated enzyme, serves as the rate limiting step in glucan biosynthesis and is an attractive target for protein engineering to increase yield. The enzyme from Thermodesulfovibrio yellowstonii (Td.y) has been successfully expressed in E. coli and a purification scheme that includes hydroxyapatite and anion exchange chromatography optimized. The inclusion of 250 mM NaCl in the running and storage buffers increased the yield and solubility (over 20 mg/mL) of the enzyme. The Td.y ADPG PPase contains a primary sequence that deviates from consensus in the conserved KRAKPAV region (amino acids 23-29), shown to be important for activity and regulation. To explore its role, the S28A enzyme was generated and characterized with respect to temperature, pH, and kinetic parameters for substrates and effectors. The S28A altered enzyme was found to have th...