title: Investigating the roles of T224 and T232 in the oxidation of cinnamaldehyde catalyzed by myxobacterial CYP260B1.
abstract: 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 CYP260B1. Following our docking studies of the aldehyde, we generated T224A and T234A mutants of CYP260B1 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.
authors.affiliation: Institut für Biochemie, Universität des Saarlandes, Saarbruecken, Germany.
authors.forename: Martin
authors.initials: M
authors.lastname: Litzenburger
authors.affiliation: Institut für Biochemie, Universität des Saarlandes, Saarbruecken, Germany.
authors.forename: Roberta
authors.initials: R
authors.lastname: Lo Izzo
authors.affiliation: Institut für Biochemie, Universität des Saarlandes, Saarbruecken, Germany.
authors.forename: Rita
authors.initials: R
authors.lastname: Bernhardt
authors.affiliation: Institut für Biochemie, Universität des Saarlandes, Saarbruecken, Germany.
authors.forename: Yogan
authors.initials: Y
authors.lastname: Khatri
mesh_terms: D000171:Acrolein / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D055162:Biocatalysis; D002934:Cinnamates / Q000378:metabolism; D018360:Crystallography, X-Ray; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000378:metabolism*; D003903:Deuterium / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000737:chemistry; D009231:Myxococcales / Q000201:enzymology*; D010084:Oxidation-Reduction; D011374:Progesterone / Q000378:metabolism; D011522:Protons; D012997:Solvents; D013056:Spectrophotometry, Ultraviolet; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D013912:Threonine / Q000378:metabolism*
chemical_list: D002934:Cinnamates; D050505:Mutant Proteins; D011522:Protons; D012997:Solvents; C029010:cinnamic acid; D013912:Threonine; D011374:Progesterone; D000171:Acrolein; D003577:Cytochrome P-450 Enzyme System; D003903:Deuterium; C012843:cinnamaldehyde
keywords: CYP260B1; Sorangium cellulosum So ce56; aldehyde oxidation; kinetic solvent isotope effect