A metabolomics analysis to define the source of heme precursors during erythropoiesis

Heme is a cofactor essential to a vast array of metabolic and regulatory pathways. Although the eight enzymatic steps of heme synthesis have been thoroughly characterized, the source of carbon for succinyl-CoA, a substrate for the first step in heme synthesis, has received only modest attention. During erythropoiesis, the demand for heme to supply hemoglobinization necessitates a robust means to replenish succinyl-CoA. To address this question we carried out a metabolomics-based approach that employed 13C labeling. These experiments demonstrated that glutamine efficiently supplies carbons for heme synthesis and that inhibition of glutamine metabolism with a pan transaminase inhibitor halts erythroid differentiation by impairing heme synthesis. Comparing activities of tricarboxcylic acid (TCA) cycle enzymes in differentiated versus undifferentiated mouse erythroleukemia cells revealed that -ketoglutarate dehydrogenase (KDH) activity increases during erythroid differentiation. Furthermore, we identified a protein-protein interaction between KDH and aminolevulinic acid synthase 2 (ALAS2), the erythroid specific form of the first enzyme in the heme synthesis pathway. This interaction increased the activity of both KDH and ALAS2 when low levels of CoA were used. Our data illustrates that there is a metabolic shift during erythroid differentiation and that this shift relies heavily upon glutamine metabolism.