Stringency of the 2-His–1-Asp Active-Site Motif in Prolyl 4-Hydroxylase

The non-heme iron(II) dioxygenase family of enzymes contain a common 2-His–1-carboxylate iron-binding motif. These enzymes catalyze a wide variety of oxidative reactions, such as the hydroxylation of aliphatic C–H bonds. Prolyl 4-hydroxylase (P4H) is an α-ketoglutarate-dependent iron(II) dioxygenase that catalyzes the post-translational hydroxylation of proline residues in protocollagen strands, stabilizing the ensuing triple helix. Human P4H residues His412, Asp414, and His483 have been identified as an iron-coordinating 2-His–1-carboxylate motif. Enzymes that catalyze oxidative halogenation do so by a mechanism similar to that of P4H. These halogenases retain the active-site histidine residues, but the carboxylate ligand is replaced with a halide ion. We replaced Asp414 of P4H with alanine (to mimic the active site of a halogenase) and with glycine. These substitutions do not, however, convert P4H into a halogenase. Moreover, the hydroxylase activity of D414A P4H cannot be rescued with small molecules. In addition, rearranging the two His and one Asp residues in the active site eliminates hydroxylase activity. Our results demonstrate a high stringency for the iron-binding residues in the P4H active site. We conclude that P4H, which catalyzes an especially demanding chemical transformation, is recalcitrant to change

The Role of Chloride in the Mechanism of O₂ Activation at the Mononuclear Nonheme Fe(II) Center of the Halogenase HctB

Mononuclear nonheme Fe­(II) (MNH) and α-ketoglutarate (α-KG) dependent halogenases activate O₂ to perform oxidative halogenations of activated and nonactivated carbon centers. While the mechanism of halide incorporation into a substrate has been investigated, the mechanism by which halogenases prevent oxidations in the absence of chloride is still obscure. Here, we characterize the impact of chloride on the metal center coordination and reactivity of the fatty acyl-halogenase HctB. Stopped-flow kinetic studies show that the oxidative transformation of the Fe­(II)-α-KG-enzyme complex is >200-fold accelerated by saturating concentrations of chloride in both the absence and presence of a covalently bound substrate. By contrast, the presence of substrate, which generally brings about O₂ activation at enzymatic MNH centers, only has an ∼10-fold effect in the absence of chloride. Circular dichroism (CD) and magnetic CD (MCD) studies demonstrate that chloride binding triggers changes in the metal center ligation: chloride binding induces the proper binding of the substrate as shown by variable-temperature, variable-field (VTVH) MCD studies of non-α-KG-containing forms and the conversion from six-coordinate (6C) to 5C/6C mixtures when α-KG is bound. In the presence of substrate, a site with square pyramidal five-coordinate (5C) geometry is observed, which is required for O₂ activation at enzymatic MNH centers. In the absence of substrate an unusual trigonal bipyramidal site is formed, which accounts for the observed slow, uncoupled reactivity. Molecular dynamics simulations suggest that the binding of chloride to the metal center of HctB leads to a conformational change in the enzyme that makes the active site more accessible to the substrate and thus facilitates the formation of the catalytically competent enzyme–substrate complex. Results are discussed in relation to other MNH dependent halogenases

Chiral Hydroxylation at the Mononuclear Nonheme Fe(II) Center of 4-(<i>S</i>) Hydroxymandelate Synthase – A Structure-Activity Relationship Analysis

<div><p>(<i>S</i>)-Hydroxymandelate synthase (Hms) is a nonheme Fe(II) dependent dioxygenase that catalyzes the oxidation of 4-hydroxyphenylpyruvate to (<i>S</i>)-4-hydroxymandelate by molecular oxygen. In this work, the substrate promiscuity of Hms is characterized in order to assess its potential for the biosynthesis of chiral α-hydroxy acids. Enzyme kinetic analyses, the characterization of product spectra, quantitative structure activity relationship (QSAR) analyses and in silico docking studies are used to characterize the impact of substrate properties on particular steps of catalysis. Hms is found to accept a range of α-oxo acids, whereby the presence of an aromatic substituent is crucial for efficient substrate turnover. A hydrophobic substrate binding pocket is identified as the likely determinant of substrate specificity. Upon introduction of a steric barrier, which is suspected to obstruct the accommodation of the aromatic ring in the hydrophobic pocket during the final hydroxylation step, the racemization of product is obtained. A steady state kinetic analysis reveals that the turnover number of Hms strongly correlates with substrate hydrophobicity. The analysis of product spectra demonstrates high regioselectivity of oxygenation and a strong coupling efficiency of C-C bond cleavage and subsequent hydroxylation for the tested substrates. Based on these findings the structural basis of enantioselectivity and enzymatic activity is discussed.</p></div

Steady state kinetic constants of <i>S. coelicolor</i> Hms for a range of aromatic 2-oxo acids and corresponding product enantiopurity.

<p>Apparent kinetic constants were determined at air saturation (20 mM Tris buffer, pH 7.5, 25°C).</p>*<p>The analytical method did not allow a more precise determination.</p

Substrate and product ligands docked into the <i>S. coelicolor</i> Hms model in silico.

<p>Several active site residues are omitted for clarity. Column 1 shows HPP (white) and (<i>S</i>)-<i>p</i>-hydroxymandelate (black), column 2 gives PP (apricot) and (<i>S</i>)-mandelate (light pink), column 3 depicts <i>p</i>-methoxy-PP (teal) and (<i>S</i>)-<i>p</i>-methoxymandelate (purple) and column 4 displays 2-oxo-4-phenylbutanoic acid (pink) and (<i>S</i>)-4-phenyllactate (light blue). Indicated distances are summarized in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068932#pone.0068932.s012" target="_blank">Table S5</a>.</p

Specific activity of <i>S. coelicolor</i> Hms for a range of aliphatic 2-oxo acids and corresponding calculated substrate log <i>D</i> values.

<p>Measurements were performed in air saturated 20 mM Tris buffer at pH 7.5 and 25°C. Values for HPP are given in comparison. The limit of detection under assay conditions was a specific rate of 10⁻⁵ s⁻¹.</p