Structural and functional studies of plant glucan phosphatases

Abstract

Glucan phosphatases (GPs) dephosphorylate starch to regulate degradation. Historically, GPs have been defined as proteins containing a dual specificity phosphatase (DSP) domain and a carbohydrate binding module (CBM). To date there are two published GPs: Starch‐Excess‐4 (SEX4) and Like‐SEX4‐1 (LSF1). Our lab recently determined the structure of SEX4. These data identified a previously unknown domain in the C‐terminus of GPs, the so‐called CT‐domain. The discovery of this domain allowed us to identify a third member of the GP family, LSF2. LSF2 contains both a DSP and CT domain, but lacks a CBM. We cloned LSF2, purified recombinant protein, and demonstrated that it has glucan phosphatase activity even though it lacks a CBM. Furthermore, SEX4 and LSF2 possess different specificities: SEX4 dephosphorylates starch preferentially at the C6 position of glucose, while LSF2 acts on the C3 position. The function of LSF1 is still unknown as it lacks phosphatase activity thus far. We are performing crystallization screenings on LSF1 and LSF2 to obtain their structures, and we are using molecular modeling of LSF1 and LSF2 to determine key residues involved in glucan binding. In addition, we aligned the known orthologs of each gene and mapped the degree of conservation on the structures to infer critical regions for GP activity. Cumulatively, these results allow us to determine the molecular mechanism of this class of enzymes.Supported by NIH grants R00NS061803, P20RR020171, and R01NS070899, and University of Kentucky College of Medicine startup funds to M.S.G.