Expanding substrate specificity of salicylate decarboxylase by site-directed mutagenesis for expansion of the entrance region connecting to the substrate access tunnel

Salicylate decarboxylase (Sdc, EC 4.1.1.91) from the yeast Trichosporon moniliiforme WU-0401 catalyzes the carboxylation of phenol to salicylic acid and is applicable to enzymatic Kolbe-Schmitt reaction. Based on the 3D-modeling of Sdc, we generated a Sdc mutant, K23A-Sdc, by site-directed mutagenesis for expansion of the entrance region connecting to the substrate access tunnel. K23A-Sdc catalyzed the carboxylation of o-cresol to 3-methyl salicylic acid (3-MS), whereas Sdc showed negligible and slight activity toward o-cresol and 3-MS, respectively. Simultaneously, K23A-Sdc showed approximately 5.8-fold more decarboxylation activity toward 3-MS than Sdc. These results clearly indicate that K23A-Sdc acquired novel substrate specificity by substitution of only one amino acid residue (23rd lysine residue to alanine residue), around the substrate entrance region but not at the active center of Sdc.