|dc.description.abstract||COG2159 of the Amidohydrolase Superfamily (AHS) is composed of a wide range of enzymes, which catalyze hydration, hydrolysis, and decarboxylation reactions. 5-Carboxyvanillate decarboxylase (LigW) belongs to COG2159 and catalyzes the conversion of 5-carboxyvanillate (5-CV) to vanillate (VAN) in the pathway for the degradation of lignin. The recombinant genes from Sphingominas paucimobilis SYK-6 (LigW) and Novosphingobium aromaticivorans DSM 12444 (LigW2) were expressed in E. coli in the presence of Mn^2+ and the purified enzymes contained 1 equivalent of Mn^2+. The kinetic constants for the decarboxylation of 5-CV are as follows: kcat = 2.0 s^-1 and kcat/Km = 4.4 x 10^4 M^-1 s^-1 for the SYK-6 LigW and kcat = 27 ± 1.0 s^-1 and kcat/Km = 1.1 x 10^4 M^-1 s^-1 for the DSM 12444 LigW2. The pH-rate profiles are bell-shaped, these results are consistent with the required deprotonation of the invariant Asp-296 and protonation of His-226 for catalysis and/or substrate binding to occur. Alterations of LigW metal ligands significantly decrease the catalytic activity with kcat/Km values at least three orders of magnitude lower than that of the wild-type enzyme. Site-directed mutagenesis of substrate binding resides substantially lower or abolish the activity in LigW. The enzyme is also inhibited by the product vanillate, 3-methoxy-5-carboxybenzoate, and 4-hydroxy-3-methoxy-5-nitrobenzoate. The latter proved to be a tight binding inhibitor of LigW and LigW2 with a Ki^app = 17 nM for each of the enzymes. LigW catalyzes the exchange of the hydrogen at C-5 of VAN with deuterium over time and the product isotope effect (PIE) in a 50:50 mixture of D2O:H2O at pD 9.0 is 4.3. The crystal structures of LigW from SYK-6 and DSM 12444 with Mn^2+ in the active site were determined to a resolution of 1.8 A (PDB id: 4ICM) and 1.5 A (PDB id: 4INF). The structure of LigW was also determined complexed with the inhibitors 5-NV, MCB, and VAN. A chemical mechanism for the decarboxylation of 5-CV to VAN by LigW has been proposed. LigW requires Mn2+ for catalysis, proton transfer to C-5 is likely rate limiting for the overall reaction and precedes the decarboxylation step. The protonation of the si-face of C5 is performed from either the hydroxyl group at C4 or the carboxylate group of Asp-296.
In addition, COG2159 γ-resorcylate decarboxylase (γ-RSD) from Polaromonas sp. JS666 catalyzes the conversion of γ-resorcylate to resorcinol this enzyme also catalyzes the decarboxylation of 2, 4, 6- trihydroxybenzoate, 2, 3- dihydroxybenzoate, and 2, 6-dihydroxy-4-methylbenzoate.||