Modulating Chemoselectivity in a Fe(II)/α-Ketoglutarate-Dependent Dioxygenase for the Oxidative Modification of a Nonproteinogenic Amino Acid

Abstract

Modification of aliphatic C-H bonds in a regio- and stereoselective manner can pose a formidable challenge as these are least reactive in organic chemistry. In this context, the use of non-heme iron and α-ketoglutarate-dependent dioxygenases (αKGDs) represents an interesting complementary tool as this enzyme family can catalyze a broad set of synthetically valuable reactions including hydroxylations, oxidations and desaturations. The consensus reaction mechanism of this enzyme family proceeds via the formation of a Fe(IV)-oxo complex capable of hydrogen atom transfer (HAT) from a sp3- hybridized substrate carbon center. The resulting substrate radical and Fe(III)-OH cofactor is 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/ α-ketoglutarate-dependent oxygenases via enzyme engineering has been mainly elusive. In this study, we therefore targeted to engineer the L-proline cis-4-hydroxylase SmP4H from Sinorhizobium meliloti for selective oxidative modification of the non-proteinogenic amino acid L-homo-phenylalanine (L-hPhe) to produce pharmacological relevant small molecule intermediates. Using structure-guided directed evolution, we improved the turnover number and apparent kcat of the hydroxylation reaction yielding the desired -hydroxylation product by approximately 10-fold and 20-fold, respectively. Notably, the introduction of only one new catalytic entity into the active site (W40Y), allowed us to re-program the natural hydroxylase to predominantly act as a desaturase, presumably through tyrosine’s capability to serve as a catalytic base in the reaction mechanism.