Defining Thermophilic Glucan Dikinases in Cyanidioschyzon merolae

Abstract

ObjectiveThe goal of this project is to elucidate the mechanism of action of starch dikinases in carbohydrate metabolism using the red alga Cyanidioschyzon merolae (Cm) as a model system.SignificanceHighly regulated reversible starch phosphorylation is key for starch metabolism in photosynthetic organisms such as plants and algae. Defining the enzymes that are involved in reversible starch phosphorylation in C. merolae will allow us to engineer strategies to harness starch production in a more efficient manner for many industrial applications.MethodsBioinformatics to identify conserved functional domains with orthologs from different kingdoms. Radiometric kinase assays to measure specificity and kinetic parameters of starch dikinases. Differential Scanning Fluorimetry (DSF) to characterize glucan binding and protein thermal stability. Cell biology techniques such as gene suppression, synchronized cultures, mRNA extraction, and immunofluorescence. Structural analysis through Small Angle X‐Ray Scattering (SAXS), protein purification, and western analysis.ResultsWe show here that C. merolae GWD shares conserved functional domains with orthologs from many different kingdoms. Validating Cm‐GWD is a functional glucan dikinase, results show robust Cm‐GWD specificity towards phosphorylating the C6 position of glucose residues. Radiometric‐ATP incorporation assays reveals that recombinant Cm‐GWD is as efficient as Arabidopsis thaliana (At)‐GWD at phosphorylating starch. Lastly, we utilize SAXS to structurally characterize each domain of the multi‐domain Cm‐GWD.ConclusionWe have defined C. merolae growth and optimal starch accumulation parameters as well as cell localization of enzymes involved in reversible starch phosphorylation. Furthermore, we have characterized gene expression of Cm‐GWD via mRNA levels during synchronous cultures. Additionally, this study exposes biochemical targets to enhance starch metabolism in planta. The biochemical properties of Cm‐GWD enable increased starch metabolism not only in C. merolae but in exogenous plants.Support or Funding InformationKSEF grants KSEF‐2268RDE‐014 and KSEF‐2971‐RDE‐017; NSF Grants IIA‐1355438 and MCB‐1252345