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Defining Thermophilic Glucan Dikinases in Cyanidioschyzon merolae
Author(s) -
Brizzee Corey O.,
Husodo Satrio,
Gentry Matthew S.
Publication year - 2016
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.30.1_supplement.1145.1
Subject(s) - biochemistry , starch , phosphorylation , biology , enzyme , chemistry , microbiology and biotechnology
Objective The 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. Significance Highly 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. Methods Bioinformatics 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. Results We 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. Conclusion We 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 Information KSEF grants KSEF‐2268RDE‐014 and KSEF‐2971‐RDE‐017; NSF Grants IIA‐1355438 and MCB‐1252345