(344h) A Kinetic Model of Plant Sphingolipid Metabolism Identifies Key Enzymes and Regulatory Interactions in the Pathway

Sphingolipids are an essential component of many eukaryotic cells including human and plant cell’s plasma membrane and endomembranes. In plants, this class of lipids plays several functional roles including providing structural integrity to the membrane, golgi trafficking, and protein organizational domains. In addition, sphingolipids have been implicated in physiological processes such as the signaling of Programmed Cell Death (PCD) and the hypersensitive response associated with plant resistance to bacterial and fungal pathogens. The metabolic pathways associated with sphingolipid biosynthesis are tightly controlled to ensure sufficient sphingolipid availability for normal cell growth. Simultaneously, metabolic controls constrain the accumulation of sphingolipid building blocks responsible for the induction of PCD until this process is required (e.g. during the pathogen triggered hypersensitive response). Recent work has shown that orosomucoid-like (ORM) proteins are involved in the regulation of multiple enzymes in the pathway. In this work, a combined computational and experimental approach was employed to mechanistically decipher the regulation of sphingolipid biosynthesis. A compartmentalized metabolic network of sphingolipid biosynthesis in Arabidopsis thaliana has been reconstructed. Flux Balance Analysis (FBA) has been used to simulate the steady-state flux distribution of the network at the measured uptake rates of the starting material sphingosine. An Ensemble Modeling (EM) framework has then been implemented to construct a kinetic model of the metabolic and regulatory network of Sphingolipid biosynthesis. This EM approach allows for the prediction of enzyme kinetics and metabolite concentrations given a metabolic network and a set of experimentally determined reaction fluxes. Incorporating experimental data from multiple cell lines, the kinetic model has been used to predict the global regulatory role in the pathway. Namely, model predictions indicate the presence of a ceramide-ORM-ceramide synthase (class II) regulatory interaction. In this scheme, ORMs activate class II ceramide synthases (LOH1/LOH3); however, upon ceramides accumulation ORMs are repressed and unable to activate CS II causing a buildup of LCBs. Furthermore, sensitivity analysis identified the crucial role of SBH in maintaining homeostasis. These results illustrate how kinetic models can be used to predict regulatory mechanisms in biochemical systems despite lack of prior knowledge on enzyme kinetics.