Molecular mechanism of glycolytic assembly on mitochondria through O-GlcNAcylation.

Abstract

Glucose is the primary fuel of cells, and its metabolism starts with the activity of the first rate-limiting enzyme Hexokinase (HK). HK1 is the dominant isoform in the brain and is mainly localized near the mitochondrial outer membrane. The positioning of HK1 on mitochondria is critical because it couples two energy generation pathways: Glycolysis and mitochondrial oxidative phosphorylation. Here, we report a new molecular mechanism that regulates HK1 activity and mitochondrial localization via the metabolic sensor enzyme O-GlcNAc transferase (OGT). OGT catalyzes a reversible post-translational modification by adding a GlcNAc sugar moiety to serine and threonine residues (O-GlcNAcylation). By combining experiments and molecular simulations, this study shows that the dynamical modification of HK1 with O-GlcNAcylation at its regulatory domain disrupts the product, G6P, from binding to this non-catalytic domain. We further demonstrate that G6P binding to the non-catalytic domain plays a role in controlling mitochondrial localization through electrostatic repulsion due to the additional negative charge from G6P. In addition, we demonstrated that O-GlcNAc modification induces a conformational change which increases mitochondrial HK1 and enhances glycolytic and mitochondrial ATP production rates. By computing binding affinities of the O-GlcNAc modified and unmodified HK1, we see that the O-GlcNAcylation decreases G6P and increases ATP binding to HK1. Furthermore, by mutating the O-GlcNAcylation site of HK1, we see a decrease in both glycolytic and mitochondrial ATP production rate and dysfunction of presynaptic vesicle releasing in neurons. Our findings may reveal key molecular pathways that couple neuronal metabolism to mitochondrial function via OGT and how their dysregulation leads to neurological disorders.