Operando ¹H nuclear magnetic resonance (NMR) activity assay for pyruvate decarboxylase

The biological conversion of pyruvate by thiamine pyrophosphate (TPP)-linked enzymes, such as pyruvate decarboxylase (Pdc), is a critical step in microbial production of biofuels such as ethanol. Traditionally, the activity of these enzymes is measured using UV-Vis spectrophotometric coupled assays. However, these assays typically only measure one chemical species. Here, we describe an Operando ¹H Nuclear Magnetic Resonance (NMR) activity assay that provides a robust, non-destructive alternative for monitoring Pdc kinetics in real time. This quantitative NMR (qNMR) method allows for the simultaneous detection and quantification of substrates, products, cofactors, and intermediates in a single experiment, bypassing the need for secondary coupling enzymes and avoiding associated kinetic artifacts. We detail the experimental design, data acquisition, and data processing necessary to implement this type of assay. By providing precise, simultaneous quantification of almost all chemical species in the reaction (pyruvate, acetaldehyde, ethanol, NAD⁺, and NADH), this NMR-based protocol offers a powerful complementary tool for the characterization and optimization of microbial biofuel production pathways.