Reference Number: 130095
Principal Investigator: Dr. Matthew Gentry
Organization: University of Kentucky
Period: 4.1.13-3.31.15
Grant Title: Glucan phosphatases link neurodegeneration and biofuels research
Biofuels research and research into the fatal neurodegenerative disease called
Lafora disease (LD) are intimately linked by a newly described family of enzymes
called glucan phosphatases. Glucans are the most abundant polymer in plants andalgae, with cellulose (β-1-4 linkages) serving as the major structural component andstarch (α-1-4 and α-1-6 linkages) as the major energy reserve. Due to its abundanceand energy-rich status, starch is a common 1st generation biofuels feedstock. Insteadof starch, humans utilize glycogen as their primary carbohydrate energy storage
molecule. Both starch and glycogen metabolism is dependent on the action of glucan
phosphatases.
Plants release the energy in starch via a recently identified
three-step process: starch phosphorylation, degradation, anddephosphorylation (Figure). Plants phosphorylate the outerstarch glucose units to make them water-soluble and enzyme
accessible so that amylases can degrade the starch into
glucose, maltose, and oligosaccharides. Following amylase
activity, the phosphate must be removed by glucan
phosphatases. This three-step cycle is repeated to allow
efficient, processive starch degradation. In the absence ofphosphorylation or dephosphorylation, the amylases are very
inefficient and plants are unable to degrade the starch that it
Starch degradation. Glucanproduces.
dikinases phosphorylate starch tofacilitate starch solubility.
In humans, glycogen synthase erroneously introduces a
Amylases release glucose.
phosphate group ~1/10,000 glucose units. The human glucan
Glucan phosphatases (GPs)starch so thephosphatase laforin removes phosphate from glycogen. In the dephosphorylate
process can repeat.
absence of laforin activity, glycogen transforms into a hyperphosphorylated, water-insoluble, starch-like Lafora body (LB). LBs are the suspectedcause of neuronal apoptosis, neurodegeneration, and eventual death of LD patients.
The objectives for this proposal are to 1) determine the structure of glucan
phosphatases and 2) define the molecular enzymology of laforin and laforin
orthologs. The methods utilized for this proposal were purification of recombinant
proteins, x-ray crystallography, mutagenesis, biochemical assays, and enzyme
kinetics. Our results allowed us to successfully complete both objectives. We
optimized protocols to produce recombinant glucan phosphatases, we establishedreproducible crystallization strategies to crystalize glucan phosphatases, and we havedefined the biochemical properties of glucan phosphatases. These results werepublished in two articles: MK Brewer et al., BMC Biochemistry, 2014; M
Raththagala et al., Molecular Cell, 2015.
While much progress has been made concerning the biology of reversible starch
phosphorylation little was known about the molecular mechanisms regulating glucan
phosphatase function. The work in this proposal addressed critical information gapsof this essential pathway. This completed work defines this novel enzyme family andimpact both biofuels research and the field of neurodegeneration.