Abstract 1588 Structure, Conformational Dynamics, and Biochemical Characterization of Starch Excess4 from Zea mays

Glucan phosphatases are members of a functionally diverse dual-specificity phosphatase family of enzymes. Plant glucan phosphatase Starch Excess4 (SEX4) reversibly binds and dephosphorylates glucans, contributing to processive starch degradation in the chloroplast at night. Despite the wealth of biochemical and structural information on SEX4, little is known about the agriculturally relevant glucan phosphatase activity. To close this gap, we explored the mechanistic enzymology of Zea mays SEX4 (ZmSEX4). Previous research shows that cereal crops Zea mays and Oryza sativa have a higher SEX4 activity than the model plant Arabidopsis thaliana, and tubers Manihot esculenta and Solanum tuberosum exhibit the lowest activity. We generated several point mutations based on the sequence alignment of SEX4 from agronomically relevant crops to determine if single-residue mutations at key binding interfaces are responsible for the observed differential activity and cooperative kinetics. The dual-specificity phosphatase domain (DSP), where the active site resides, is highly conserved across all species, so mutations with the potential for kinetic effects were chosen in the carbohydrate-binding module (CBM). We took special interest in lysine residues in the CBM of SEX4 that were not conserved in ZmSEX4. We hypothesized that the potential changes in activity may be due to a non-conserved electrostatic interaction. The biochemical data for mutant proteins suggest ways to improve catalysis for potential biotechnological applications, notably in that of industrial starch degradation. The crystal structure of ZmSEX4 wild-type was determined at a resolution of 2.4 Å. Additionally, hydrogen-deuterium exchange mass spectrometry was performed to extrapolate the conformational dynamics of the protein upon carbohydrate binding. This work was supported by the National Science Foundation award MCB-2012074.