A Combined Experimental-Theoretical Study of the LigW-Catalyzed Decarboxylation of 5‑Carboxyvanillate in the Metabolic Pathway for Lignin Degradation

Although it is a member of the amidohydrolase superfamily, LigW catalyzes the nonoxidative decarboxylation of 5-carboxyvanillate to form vanillate in the metabolic pathway for bacterial lignin degradation. We now show that membrane inlet mass spectrometry (MIMS) can be used to measure transient CO₂ concentrations in real time, thereby permitting us to establish that C–C bond cleavage proceeds to give CO₂ rather than HCO₃^– as the initial product in the LigW-catalyzed reaction. Thus, incubation of LigW at pH 7.0 with the substrate 5-carboxyvanillate results in an initial burst of CO₂ formation that gradually decreases to an equilibrium value as CO₂ is nonenzymatically hydrated to HCO₃^–. The burst of CO₂ is completely eliminated with the simultaneous addition of substrate and excess carbonic anhydrase to the enzyme, demonstrating that CO₂ is the initial reaction product. This finding is fully consistent with the results of density functional theory calculations, which also provide support for a mechanism in which protonation of the C5 carbon takes place prior to C–C bond cleavage. The calculated barrier of 16.8 kcal/mol for the rate-limiting step, the formation of the C5-protonated intermediate, compares well with the observed k_cat value of 27 s^–1 for Sphingomonas paucimobilis LigW, which corresponds to an energy barrier of ∼16 kcal/mol. The MIMS-based strategy is superior to alternate methods of establishing whether CO₂ or HCO₃^– is the initial reaction product, such as the use of pH-dependent dyes to monitor very small changes in solution pH. Moreover, the MIMS-based assay is generally applicable to studies of all enzymes that produce and/or consume small-molecule, neutral gases.