title: Structural, functional, and spectroscopic characterization of the substrate scope of the novel nitrating cytochrome P450 TxtE.
abstract: A novel cytochrome P450 enzyme, TxtE, was recently shown to catalyze the direct aromatic nitration of L-tryptophan. This unique chemistry inspired us to ask whether TxtE could serve as a platform for engineering new nitration biocatalysts to replace current harsh synthetic methods. As a first step toward this goal, and to better understand the wild-type enzyme, we obtained high-resolution structures of TxtE in its substrate-free and substrate-bound forms. We also screened a library of substrate analogues for spectroscopic indicators of binding and for production of nitrated products. From these results, we found that the wild-type enzyme accepts moderate decoration of the indole ring, but the amino acid moiety is crucial for binding and correct positioning of the substrate and therefore less amenable to modification. A nitrogen atom is essential for catalysis, and a carbonyl must be present to recruit the αB'1 helix of the protein to seal the binding pocket.
authors.affiliation: Division of Chemistry and Chemical Engineering, California Institute of Technology, MC 210-41 Pasadena, CA (USA).
authors.forename: Sheel C
authors.initials: SC
authors.lastname: Dodani
authors.forename: Jackson K B
authors.initials: JK
authors.lastname: Cahn
authors.forename: Tillmann
authors.initials: T
authors.lastname: Heinisch
authors.forename: Sabine
authors.initials: S
authors.lastname: Brinkmann-Chen
authors.forename: John A
authors.initials: JA
authors.lastname: McIntosh
authors.forename: Frances H
authors.initials: FH
authors.lastname: Arnold
mesh_terms: D001665:Binding Sites; D055162:Biocatalysis; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D015394:Molecular Structure; D009566:Nitrates / Q000737:chemistry / Q000378:metabolism*; D013056:Spectrophotometry, Ultraviolet; D013379:Substrate Specificity
chemical_list: D009566:Nitrates; D003577:Cytochrome P-450 Enzyme System
keywords: aromatic nitration; crystal-structure determination; cytochromes; enzyme catalysis