A Combined Experimental-Theoretical Study of the LigW-Catalyzed Decarboxylation of 5-Carboxyvanillate in the Metabolic Pathway for Lignin Degradation Academic Article uri icon


  • 2017 American Chemical Society. 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 kcat 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.

published proceedings

  • ACS Catalysis

altmetric score

  • 0.5

author list (cited authors)

  • Sheng, X., Zhu, W., Huddleston, J., Xiang, D. F., Raushel, F. M., Richards, N., & Himo, F.

citation count

  • 25

complete list of authors

  • Sheng, Xiang||Zhu, Wen||Huddleston, Jamison||Xiang, Dao Fen||Raushel, Frank M||Richards, Nigel GJ||Himo, Fahmi

publication date

  • June 2017