Substrate Distortion and the Catalytic Reaction Mechanism of 5‑Carboxyvanillate Decarboxylase

5-Carboxyvanillate decarboxylase (LigW) catalyzes the conversion of 5-carboxyvanillate to vanillate in the biochemical pathway for the degradation of lignin. This enzyme was shown to require Mn<sup>2+</sup> for catalytic activity and the kinetic constants for the decarboxylation of 5-carboxyvanillate by the enzymes from <i>Sphingomonas paucimobilis</i> SYK-6 (<i>k</i><sub>cat</sub> = 2.2 s<sup>–1</sup> and <i>k</i><sub>cat</sub>/<i>K</i><sub>m</sub> = 4.0 × 10<sup>4</sup> M<sup>–1</sup> s<sup>–1</sup>) and <i>Novosphingobium aromaticivorans</i> (<i>k</i><sub>cat</sub> = 27 s<sup>–1</sup> and <i>k</i><sub>cat</sub>/<i>K</i><sub>m</sub> = 1.1 × 10<sup>5</sup> M<sup>–1</sup> s<sup>–1</sup>) were determined. The three-dimensional structures of both enzymes were determined in the presence and absence of ligands bound in the active site. The structure of LigW from <i>N. aromaticivorans</i>, bound with the substrate analogue, 5-nitrovanillate (<i>K</i><sub>d</sub> = 5.0 nM), was determined to a resolution of 1.07 Å. The structure of this complex shows a remarkable enzyme-induced distortion of the nitro-substituent out of the plane of the phenyl ring by approximately 23°. A chemical reaction mechanism for the decarboxylation of 5-carboxyvanillate by LigW was proposed on the basis of the high resolution X-ray structures determined in the presence ligands bound in the active site, mutation of active site residues, and the magnitude of the product isotope effect determined in a mixture of H<sub>2</sub>O and D<sub>2</sub>O. In the proposed reaction mechanism the enzyme facilitates the transfer of a proton to C5 of the substrate prior to the decarboxylation step.