The carbon starvation-induced protein D (CsiD) is a recently characterized iron(II)/α-ketoglutarate-dependent oxygenase that activates a glutarate molecule as substrate at the C2 position to exclusively produce (S)-2-hydrox-yglutarate products. This selective hydroxylation reaction by CsiD is an important component of the lysine biodegradation pathway in Escherichia coli; however, little is known on the details and the origin of the selectivity of the reaction. So far, experimental studies failed to trap and characterize any short-lived catalytic cycle intermediates. As no computational studies have been reported on this enzyme either, we decided to investigate the chemical reaction mechanism of glutarate activation by an iron(IV)-oxo model of the CsiD enzyme. In this work, we present a density functional theory study on a large active site cluster model of CsiD and investigate the glutarate hydroxylation pathways by a high-valent iron(IV)-oxo species leading to (S)-2–hydroxyglutarate, (R)-2-hydroxyglutarate and 3-hydroxyglutarate. In agreement with experimental observation, the favorable product channel leads to pro-S C2H hydrogen atom abstraction to form (S)-2-hydroxyglutarate. The reaction is stepwise with a hydrogen atom abstraction by an iron(IV)-oxo species followed by OH rebound from a radical intermediate. The work presented in this paper shows that despite the fact that the CH bond strengths at the C2 and C3 positions of glutarate are similar in the gas-phase, substrate binding and positioning guides the reaction to an enan-tioselective reaction process by destabilizing the hydrogen atom abstraction pathways for the pro-R C2H and C3H posi-tions. Our studies predict the chemical properties of the iron(IV)-oxo species, and its rate-constants with glutarate substrate and its deuterated form. Moreover, the work shows little protein motions during the catalytic process, while substrate entrance into the substrate binding pocket appears to be guided by three active site arginine residues that position the substrate for pro-S C2H hydrogen atom abstraction.
|Publication status||Accepted/In press - 16 Mar 2021|