Date Available

6-9-2016

Year of Publication

2014

Document Type

Doctoral Dissertation

Degree Name

Doctor of Philosophy (PhD)

College

Medicine

Department/School/Program

Molecular and Cellular Biochemistry

Advisor

Dr. Matthew S. Gentry

Co-Director of Graduate Studies

Dr. David W. Rodgers

Abstract

Starch is a water-insoluble glucose biopolymer used as an energy cache in plants and is synthesized and degraded in a diurnal cycle. Reversible phosphorylation of starch granules regulates the solubility and, consequentially, the bioavailability of starch glucans to degradative enzymes. Glucan phosphatases release phosphate from starch glucans and their activity is essential to the proper diurnal metabolism of starch. Previously, the structural basis of glucan phosphatase activity was entirely unknown. The work in this dissertation outlines the structural mechanism of activity of two plant glucan phosphatases called Starch EXcess4 (SEX4) and Like Sex Four2 (LSF2). The crystal structures of SEX4 and LSF2 were determined with and without phosphoglucan ligands bound, revealing the basis of their interaction with an endogenous substrate. The data show that SEX4 and LSF2 interact with starch glucans via distinctive mechanisms. SEX4 binds glucan chains via an aromatic-rich pocket spanning its Carbohydrate Binding Module (CBM) and catalytic Dual Specificity Phosphatase (DSP) domains. Conversely, LSF2 lacks a CBM and, instead, binds glucans at two non-catalytic surface-binding sites that are located distally from its active site. In addition, it was previously reported that SEX4 and LSF2 act upon distinct phospho-glucan substrates: SEX4 preferentially dephosphorylates the C6-position of starch glucans and LSF2 exclusively dephosphorylates the C3- position. The data herein reveal that SEX4 and LSF2 contain differences in their active site topology that serve to position the glucan chain in opposite orientations, therefore accounting for the differences in substrate specificity. Using these insights, SEX4 was engineered with reversed substrate specificity, i.e. preferential C3-specific activity. Previous work has established the interaction between phosphatases and protein, lipid, and nucleic acids; however, the current study represents the first insights into phosphatase interaction with carbohydrate substrates. In addition, the insights gained provide a model that will be used in future studies with the mammalian glucan phosphatase laforin, which is linked to neurodegeneration and the fatal epileptic disorder Laforaʼs Disease.

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