Mutational analysis of phenolic acid decarboxylase from Bacillus subtilis (BsPAD), which converts bio-derived phenolic acids to styrene derivativesElectronic supplementary information (ESI) available: Table containing sequences of primers for PCR and PCR-based site-directed mutagenesis; Table of Data Collection and Refinement statistics for Y19A mutant of BsPAD in complex with CA; Figure showing graph of HPLC conversions of ferulate versus [ferulate] for each variant. See DOI: 10.1039/c2cy20015e

• Main Authors: Frank, Annika, Eborall, William, Hyde, Ralph, Hart, Sam, Turkenburg, Johan P, Grogan, Gideon
• Format: Journal Article
• Published: 16.07.2012
• DOI: 10.1039/c2cy20015e
• ISSN: 2044-4753

Phenolic acid decarboxylase from Bacillus subtilis ( Bs PAD) catalyses the decarboxylation of phenolic acids such as coumaric acid to give vinyl phenols, which are of interest as possible polymer precursors and flavour/fragrance compounds. The structure of the Tyr19Ala mutant of Bs PAD has been solved in complex with coumaric acid. In the active site, the substrate carboxylate is bound by Tyr11 and Tyr13, and the phenolic hydroxyl by the NE atom of Arg41. A comparison of the mutant complex with the wild-type apoenzyme reveals that the β1-β2 loop, running from Tyr11 to Ala19, closes over the active site in the presence of substrate, shielding it from bulk solvent. The complex structure, in conjunction with an activity study of point mutants of Bs PAD, provides support for a mechanism for PADs, proposed by Mancheño and co-workers for the homologue from Lactobacillus plantarum [ Proteins , 2010, 78 , 1662-1676]. In this mechanism, a quinone methide intermediate results from deprotonation of the phenolic hydroxyl of the substrate by Glu64, assisted by Arg41. Decarboxylation of the substrate is effected through binding of the carboxylate by Tyr11 and Tyr13, the latter being brought into contact with the substrate as a result of the movement of the β1-β2 loop on substrate binding. Mutational analysis, coupled with a complex structure, reveals catalytic roles for Tyr11 and Tyr13 in carboxylate recognition and for Arg41 in binding of the phenolic hydroxyl of the substrate.