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Publication type: Article in scientific journal
Type of review: Peer review (publication)
Title: Modulating chemoselectivity in a Fe(II)/α-ketoglutarate-dependent dioxygenase for the oxidative modification of a non-proteinogenic amino acid
Authors: Meyer, Fabian
Frey, Raphael
Ligibel, Mathieu
Sager, Emine
Schroer, Kirsten
Snajdrova, Radka
Buller, Rebecca
et. al: No
DOI: 10.1021/acscatal.1c00678
Published in: ACS Catalysis
Volume(Issue): 2021
Issue: 11
Issue Date: 10-May-2021
Publisher / Ed. Institution: American Chemical Society
ISSN: 2155-5435
Language: English
Subject (DDC): 660.6: Biotechnology
Abstract: Modification of aliphatic C–H bonds in a regio- and stereoselective manner can pose a formidable challenge in organic chemistry. In this context, the use of nonheme iron and α-ketoglutarate-dependent dioxygenases (αKGDs) represents an interesting tool for C–H activation as this enzyme family can catalyze a broad set of synthetically valuable reactions including hydroxylations, oxidations, and desaturations. The consensus reaction mechanism of αKGDs proceeds via the formation of a Fe(IV)-oxo complex capable of hydrogen atom transfer (HAT) from an sp3-hybridized substrate carbon center. The resulting substrate radical and Fe(III)–OH cofactor are considered to be the branch point toward the possible reaction outcomes which are determined by the enzyme’s active site architecture. To date, the modulation of the reaction fate in Fe(II)/α-ketoglutarate-dependent dioxygenases via enzyme engineering has been mainly elusive. In this study, we therefore set out to engineer the l-proline cis-4-hydroxylase SmP4H from Sinorhizobium meliloti for selective oxidative modifications of the nonproteinogenic amino acid l-homophenylalanine (l-hPhe) to produce pharmacological relevant small molecule intermediates. Using structure-guided directed evolution, we improved the total turnover number, the kcat, as well as the kcat/Km of the hydroxylation reaction yielding the desired γ-hydroxylation product by approximately 10-fold, >100-fold, and >300-fold, respectively. Notably, the exchange of only one amino acid in the active site (W40Y) allowed us to reprogram the natural hydroxylase to predominantly act as a desaturase, presumably through tyrosine’s capability to serve as a catalytic entity in the reaction mechanism. An investigation of the substrate scope revealed additional acceptance of the noncanonical amino acids l-homotyrosine and (S)-α-amino-3,4-dichlorobenzenebutanoic acid by SmP4H variants.
Fulltext version: Accepted version
License (according to publishing contract): Licence according to publishing contract
Restricted until: 2022-05-10
Departement: Life Sciences and Facility Management
Organisational Unit: Institute of Chemistry and Biotechnology (ICBT)
Appears in collections:Publikationen Life Sciences und Facility Management

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