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Investigating the roles of T224 and T232 in the oxidation of cinnamaldehyde catalyzed by myxobacterial CYP 260B1
Author(s) -
Litzenburger Martin,
Lo Izzo Roberta,
Bernhardt Rita,
Khatri Yogan
Publication year - 2017
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1002/1873-3468.12519
Subject(s) - chemistry , aldehyde , cinnamaldehyde , aldehyde oxidase , substrate (aquarium) , nucleophile , catalysis , stereochemistry , mutagenesis , combinatorial chemistry , biochemistry , mutant , enzyme , biology , ecology , gene , xanthine oxidase
Although the oxidation of aldehydes to carboxylic acids is mainly catalyzed by aldehyde dehydrogenases in nature, cytochromes P450 are also able to perform such reactions. In this study, we demonstrate the oxidation of cinnamaldehyde to cinnamic acid by the myxobacterial CYP 260B1. Following our docking studies of the aldehyde, we generated T224A and T234A mutants of CYP 260B1 by site‐directed mutagenesis to disrupt the substrate positioning and proton delivery, respectively. Furthermore, we used the kinetic solvent isotope effect on the steady‐state turnover of the substrate to investigate the reactive intermediate capable of performing the catalysis. Our results suggest that the aldehyde oxidation occurs via a nucleophilic attack of the ferric peroxoanion.