To generate the [3,O-D2]malate sample for the pulse sequence optimization experiments, 300 mM disodium fumarate was dissolved in D2O. Subsequently, 25 μL of fumarase was added to this sample to catalyse the formation of [3,O-D2]malate. This sample was kept in a 5 mm NMR tube overnight at room temperature for the enzyme to degrade and used without further alteration.