AUTHOR=Santos Sonia F. G. , Bommareddy Rajesh Reddy , Black Gary W. , Singh Warispreet 

TITLE=Impact of the T296S mutation in P450 GcoA for aryl-O-demethylation: a QM/MM study

JOURNAL=Frontiers in Chemistry

VOLUME=Volume 11 - 2023

YEAR=2024

URL=https://www.frontiersin.org/journals/chemistry/articles/10.3389/fchem.2023.1327398

DOI=10.3389/fchem.2023.1327398

ISSN=2296-2646

ABSTRACT=Lignin, a complex plant cell wall component, holds promise as a renewable aromatic carbon feedstock. p-Vanillin is a key product from lignin depolymerization and a precursor of protocatechuic acid (PCA) that has a tremendous potential for biofuel production. While the GcoAB enzyme, native to Amycolatopsis sp., naturally catalyses aryl-O-demethylation toward Guaiacol, recent research introduced a single mutation, T296S, into the GcoAP450 enzyme, enabling it to catalyse aryl-O-demethylation of p-vanillin. This structural modification increases the efficiency of GcoAP450 for natural substrate while being active for p-Vanillin. This study reveals the increased flexibility of p-Vanillin and its ability to adapt a favorable conformation by aligning the methoxy group in close vicinity of Fe(IV)=O of Cpd I in the active site of T296S variant. The QMMM calculations in accordance with the experimental data validated the rate limiting step for the oxidation of p-Vanillin is hydrogen atom abstraction and provided the detailed geometric structure of stationary and saddle points for the oxidation of p-Vanillin.