Glutarate Hydroxylation by the Carbon Starvation-Induced Protein D: A Computational Study into the Stereo- and Regioselectivities of the Reaction

Abstract

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-hydroxyglutarate 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 C2–H 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 C–H bond strengths at the C2 and C3 positions of glutarate are similar in the gas phase, substrate binding and positioning guide the reaction to an enantioselective reaction process by destabilizing the hydrogen atom abstraction pathways for the pro-R C2–H and C3–H positions. Our studies predict the chemical properties of the iron­(IV)-oxo species and its rate constants with glutarate and deuterated-glutarate. Moreover, the work shows little protein motions during the catalytic process, while the substrate entrance into the substrate binding pocket appears to be guided by three active site arginine residues that position the substrate for pro-S C2–H hydrogen atom abstraction. Finally, the calculations show that irrespective of the position of the substrate and what C-H bond is closest to the metal center, the lowest energy pathway is for a selective pro-S C2–H hydrogen atom abstraction.

Structural Insight into the Catalytic Mechanism of the Endoperoxide Synthase FtmOx1

“…In an alternative pathway, the Fe III −OH species may undergo the OH‐rebound to the C21 radical site via a slightly larger barrier of 5.2 kcal mol −1 , affording to the hydroxylated substrate which is 27.6 kcal mol −1 more stable than the intermediate IC1 (Figure S13). The calculated rebound barrier is in line with previous studies [60–73] . The hydroxylated species would lead to the side product 8 through elimination (refer to Scheme 3), as observed in experiments.…”

“…Thec alculated rebound barrier is in line with previous studies. [60][61][62][63][64][65][66][67][68][69][70][71][72][73] The hydroxylated species would lead to the side product 8 through elimination (refer to Scheme 3), as observed in experiments. Clearly,t he dioxygen attack is kinetically favorable over the rebound pathway,w hile the rebound pathway is more thermodynamically favorable.Inthe situation of low concen- S3 and Figure S15).…”

Density Functional Theory Study into the Reaction Mechanism of Isonitrile Biosynthesis by the Nonheme Iron Enzyme ScoE

“…The Gaussian-09 software package was used for all quantum chemical calculations discussed here [55]. Following previous experience with cluster models of nonheme iron dioxygenases [53,56,57], we utilized the unrestricted B3LYP density functional method [58,59] in combination with a LANL2DZ (with electron core potential) on iron and 6-31G on the rest of the atoms: basis set BS1 [60,61]. To correct the energetics, single point calculations with the LACV3P + (with electron core potential) on iron and 6-311 + G* on the rest of the atoms were performed (basis set BS2).…”

Inspiration from Nature: Influence of Engineered Ligand Scaffolds and Auxiliary Factors on the Reactivity of Biomimetic Oxidants

“…In addition, computational studies on the biosynthesis nonheme iron dioxygenase HygX identified a positively charged active site Lys residue that through charge-dipole interactions influences reactivity patterns and guides the selectivity of the reaction . However, in the analogous enzymes viomycin biosynthesis enzyme (VioL), the 2-(trimethylammonio)­ethylphosphonate dioxygenase (TmpA) and the carbon starvation-induced protein D (CsiD), a combination of positively and negatively charged residues arranged local dipole moments in such a way that an otherwise unfavorable reaction pathway was stabilized. − These long-range interactions were shown to affect redox potentials and bond strengths and hence influence the catalytic cycle and enzymatic turnover. To understand the technical details of second- and first-coordination sphere effects of enzymes, many inorganic chemists have made links of enzymatic structures with synthetic (biomimetic) models.…”