Substrate Distortion and the Catalytic Reaction Mechanism of 5-Carboxyvanillate Decarboxylase
Fedorov, Alexander A.
Bonanno, Jeffrey B.
Fedorov, Elena V.
Seidel, Ronald D.
Richards, Nigel G. J.
Almo, Steven C.
Raushel, Frank M.
- Publisher: American Chemical Society
Journal of the American Chemical Society,
(issn: 0002-7863, eissn: 1520-5126)
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 Mn2+ for catalytic activity and the kinetic constants for the decarboxylation of 5-carboxyvanillate by the enzymes from Sphingomonas paucimobilis SYK-6 (kcat = 2.2 s–1 and kcat/Km = 4.0 × 104 M–1 s–1) and Novosphingobium aromaticivorans (kcat = 27 s–1 and kcat/Km = 1.1 × 105 M–1 s–1) 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 N. aromaticivorans, bound with the substrate analogue, 5-nitrovanillate (Kd = 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 H2O and D2O. In the proposed reaction mechanism the enzyme facilitates the transfer of a proton to C5 of the substrate prior to the decarboxylation step.