Abstract 1712 Unveiling Key Sites and Functional Impact of Putative Phosphorylation on Human Cytosolic Malate Dehydrogenase
Year: 2024
Type: conference-abstract
Abstract: Phosphorylation serves as a critical regulatory mechanism governing numerous metabolic processes. Despite the identification of over 22 phosphorylation sites on human cytosolic malate dehydrogenase (hMDH1) and numerous predicted phospho-sites in various databases, the details surrounding the phosphorylation events targeting hMDH1 and their impact have remained largely unexplored. In this study, we investigate potential phosphorylation sites on cytosolic MDH and their functional implications, employing a comprehensive approach involving enzyme kinetics, size exclusion chromatography, melting point (Tm) analysis, and small-angle X-ray scattering (SAXS) to unravel the intricacies of MDH regulation. Several key enzymes involved in intermediate carbohydrate metabolism, such as fructokinase, pyruvate dehydrogenase, and pyruvate kinase, undergo phosphorylation. Notably, phosphorylation significantly alters the quaternary structure and activity of closely related lactate dehydrogenase, impacting cell growth and tumor development. Hence, it is intriguing to explore whether MDH plays a similar role in cancer progression and metabolic diseases by influencing metabolism. Our focus is on uncovering the spatial relationships of phosphorylation sites with key functional domains of MDH and predicting interaction interfaces with subunits or metabolic partners, including phosphoenolpyruvate carboxykinase, aspartate transaminase, ATP citrate lyase, and malic enzyme. Through the expression and purification of wild-type his-tagged recombinant hMDH1V3 and Ser/Thr to Ala mutants serving as phosphomimics at key sites (S42, S260, S261, and S264), we reveal distinct effects on enzymatic activity. Substitution of hMDH1V3 S264D with a phosphomimetic results in a threefold increase in specific activity, while hMDH1V3 S42D leads to a 2.5-fold activation. Conversely, mutagenesis targeting S260 causes a 90-fold inhibition, and S261 results in a 2.5-fold inhibition, highlighting differences in potential potent inhibitory effects at separate sites. Overall, our study significantly contributes to the expanding knowledge of cytosolic MDH phosphorylation, emphasizing the importance of site-specific phosphorylation in modulating enzymatic activity. These insights provide a deeper understanding of the regulatory network governing cellular metabolism, opening avenues for further exploration into the allosteric regulation of metabolon partners associated with cytosolic MDH. This work was supported in part by the NSF MCB RUI-1932755. This work was also conducted at the Advanced Light Source (ALS), on behalf of the Department of Energy, Office of Basic Energy Sciences, through the Integrated Diffraction Analysis Technologies (IDAT) program.
Source: Journal of Biological Chemistry
Authors: Sophie Riley, Angelina Sardelli, Angela Kayll, Colby Thompson, Christopher Berndsen, Joseph Provost
Institution: University of San Diego
Language: English
Subfield: Materials Chemistry
Field: Materials Science
Domain: Physical Sciences
Open Access status: gold
Estimated APC paid: $2,800
OpenAlex ID: W4393139008