Thermodynamics of Iron(II) and Substrate Binding to the Ethylene-Forming Enzyme.

The ethylene-forming enzyme (EFE), like many other 2-oxoglutarate (2OG)-dependent nonheme iron(II) oxygenases, catalyzes the oxidative decarboxylation of 2OG to succinate and CO2 to generate a highly reactive iron species that hydroxylates a specific alkane C-H bond, in this case targeting l-arginine (Arg) for hydroxylation. However, the prominently observed reactivity of EFE is the transformation of 2OG into ethylene and three molecules of CO2. Crystallographic and biochemical studies have led to several proposed mechanisms for this 2-fold reactivity, but the detailed reaction steps are still obscure. Here, the thermodynamics associated with iron(II), 2OG, and Arg binding to EFE are studied using calorimetry (isothermal titration calorimetry and differential scanning calorimetry) to gain insight into how these binding equilibria organize the active site of EFE, which may have an impact on the O2 activation pathways observed in this system. Calorimetric data show that the addition of iron(II), Arg, and 2OG increases the stability over that of the apoenzyme, and there is distinctive cooperativity between substrate and cofactor binding. The energetics of binding of 2OG to Fe·EFE are consistent with a unique monodentate binding mode, which is different than the prototypical 2OG coordination mode in other 2OG-dependent oxygenases. This difference in the pre-O2 activation equilibria may be important for supporting the alternative ethylene-forming chemistry of EFE.

The Irving-Williams series and the 2-His-1-carboxylate facial triad: a thermodynamic study of Mn2+, Fe2+, and Co2+ binding to taurine/α-ketoglutarate dioxygenase (TauD).

Taurine/α-ketoglutarate (αKG) dioxygenase (TauD) is an E. coli nonheme Fe2+- and αKG-dependent metalloenzyme that catalyzes the hydroxylation of taurine, leading to the production of sulfite. The metal-dependent active site in TauD is formed by two histidine and one aspartate that coordinating to one face of an octahedral coordination geometry, known as the 2-His-1-carboxylate facial triad. This motif is found in many nonheme Fe2+ proteins, but there is limited information on the thermodynamic parameters that govern metal-ion binding to this site. Here, we report data from calorimetry and related biophysical techniques to generate complete thermodynamic profiles of Mn2+ and Co2+ binding to TauD, and these values are compared to the Fe2+ data reported earlier Henderson et al. (Inorg Chem 54: 2278-2283, 2015). The buffer-independent binding constants (K) were measured to be 1.6 × 106, 2.4 × 107, and 1.7 × 109, for Mn2+, Fe2+, and Co2+, respectively. The corresponding ΔG° values were calculated to be - 8.4, - 10.1, and - 12.5 kcal/mol, respectively. The metal-binding enthalpy changes (ΔH) for these binding events are - 11.1 (± 0.1), - 12.2 (± 0.1), and - 16.0 (± 0.6) kcal/mol, respectively. These data are fully consistent with the Irving-Williams series, which show an increasing affinity for transition metal ions across the periodic table. It appears that the periodic increase in affinity, however, is a result of a complicated summation of enthalpy terms (including favorable metal-ion coordination processes and unfavorable ionization events) and related entropy terms.

Structures and Mechanisms of the Non-Heme Fe(II)- and 2-Oxoglutarate-Dependent Ethylene-Forming Enzyme: Substrate Binding Creates a Twist.

The ethylene-forming enzyme (EFE) from Pseudomonas syringae pv. phaseolicola PK2 is a member of the mononuclear nonheme Fe(II)- and 2-oxoglutarate (2OG)-dependent oxygenase superfamily. EFE converts 2OG into ethylene plus three CO2 molecules while also catalyzing the C5 hydroxylation of l-arginine (l-Arg) driven by the oxidative decarboxylation of 2OG to form succinate and CO2. Here we report 11 X-ray crystal structures of EFE that provide insight into the mechanisms of these two reactions. Binding of 2OG in the absence of l-Arg resulted in predominantly monodentate metal coordination, distinct from the typical bidentate metal-binding species observed in other family members. Subsequent addition of l-Arg resulted in compression of the active site, a conformational change of the carboxylate side chain metal ligand to allow for hydrogen bonding with the substrate, and creation of a twisted peptide bond involving this carboxylate and the following tyrosine residue. A reconfiguration of 2OG achieves bidentate metal coordination. The dioxygen binding site is located on the metal face opposite to that facing l-Arg, thus requiring reorientation of the generated ferryl species to catalyze l-Arg hydroxylation. Notably, a phenylalanyl side chain pointing toward the metal may hinder such a ferryl flip and promote ethylene formation. Extensive site-directed mutagenesis studies supported the importance of this phenylalanine and confirmed the essential residues used for substrate binding and catalysis. The structural and functional characterization described here suggests that conversion of 2OG to ethylene, atypical among Fe(II)/2OG oxygenases, is facilitated by the binding of l-Arg which leads to an altered positioning of the carboxylate metal ligand, a resulting twisted peptide bond, and the off-line geometry for dioxygen coordination.

Global stability of an α-ketoglutarate-dependent dioxygenase (TauD) and its related complexes.

BACKGROUND: TauD is a nonheme iron(II) and α-ketoglutarate (αKG) dependent dioxygenase, and a member of a broader family of enzymes that oxidatively decarboxylate αKG to succinate and carbon dioxide thereby activating O2 to perform a range of oxidation reactions. However before O2 activation can occur, these enzymes bind both substrate and cofactor in an effective manner. Here the thermodynamics associated with substrate and cofactor binding to FeTauD are explored. METHODS: Thermal denaturation of TauD and its enzyme-taurine, enzyme-αKG, and enzyme-taurine-αKG complexes are explored using circular dichroism (CD) spectroscopy and differential scanning calorimetry (DSC). RESULTS: Taurine binding is endothermic (+26kcal/mol) and entropically driven that includes burial of hydrophobic surfaces to close the lid domain. Binding of αKG is enthalpically favorable and shows cooperativity with taurine binding, where the change in enthalpy associated with αKG binding (δΔHcal) increases from -30.1kcal/mol when binding to FeTauD to -65.2kcal/mol when binding to the FeTauD-taurine complex. CONCLUSIONS: The intermolecular interactions that govern taurine and αKG binding impact the global stability of TauD and its complexes, with clear and dramatic cooperativity between substrate and cofactor. GENERAL SIGNIFICANCE: Thermal denaturation of TauD and its enzyme-taurine, enzyme-αKG, and enzyme-taurine-αKG complexes each exhibited increased temperature stability over the free enzyme. Through deconvolution of the energetic profiles for all species studied, a thermodynamic cycle was generated that shows significant cooperativity between substrate and cofactor binding which continues to clarity the events leading up O2 activation.

A cobalt-containing eukaryotic nitrile hydratase.

Nitrile hydratase (NHase), an industrially important enzyme that catalyzes the hydration of nitriles to their corresponding amides, has only been characterized from prokaryotic microbes. The putative NHase from the eukaryotic unicellular choanoflagellate organism Monosiga brevicollis (MbNHase) was heterologously expressed in Escherichia coli. The resulting enzyme expressed as a single polypeptide with fused α- and ß-subunits linked by a seventeen-histidine region. Size-exclusion chromatography indicated that MbNHase exists primarily as an (αß)2 homodimer in solution, analogous to the α2ß2 homotetramer architecture observed for prokaryotic NHases. The NHase enzyme contained its full complement of Co(III) and was fully functional without the co-expression of an activator protein or E. coli GroES/EL molecular chaperones. The homology model of MbNHase was developed identifying Cys400, Cys403, and Cys405 as active site ligands. The results presented here provide the first experimental data for a mature and active eukaryotic NHase with fused subunits. Since this new member of the NHase family is expressed from a single gene without the requirement of an activator protein, it represents an alternative biocatalyst for industrial syntheses of important amide compounds.

Biochemical and Spectroscopic Characterization of the Non-Heme Fe(II)- and 2-Oxoglutarate-Dependent Ethylene-Forming Enzyme from Pseudomonas syringae pv. phaseolicola PK2.

The ethylene-forming enzyme (EFE) from Pseudomonas syringae pv. phaseolicola PK2 is a member of the mononuclear non-heme Fe(II)- and 2-oxoglutarate (2OG)-dependent oxygenase superfamily. This enzyme is reported to simultaneously catalyze the conversion of 2OG into ethylene and three CO2 molecules and the Cδ hydroxylation of l-arginine (l-Arg) while oxidatively decarboxylating 2OG to form succinate and carbon dioxide. A new plasmid construct for expression in recombinant Escherichia coli cells allowed for the purification of large amounts of EFE with activity greater than that previously recorded. A variety of assays were used to quantify and confirm the identity of the proposed products, including the first experimental demonstration of l-Δ1-pyrroline-5-carboxylate and guanidine derived from 5-hydroxyarginine. Selected l-Arg derivatives could induce ethylene formation without undergoing hydroxylation, demonstrating that ethylene production and l-Arg hydroxylation activities are not linked. Similarly, EFE utilizes the alternative α-keto acid 2-oxoadipate as a cosubstrate (forming glutaric acid) during the hydroxylation of l-Arg, with this reaction unlinked from ethylene formation. Kinetic constants were determined for both ethylene formation and l-Arg hydroxylation reactions. Anaerobic UV-visible difference spectra were used to monitor the binding of Fe(II) and substrates to the enzyme. On the basis of our results and what is generally known about EFE and Fe(II)- and 2OG-dependent oxygenases, an updated model for the reaction mechanism is presented.

Catalytic Mechanisms of Fe(II)- and 2-Oxoglutarate-dependent Oxygenases.

Mononuclear non-heme Fe(II)- and 2-oxoglutarate (2OG)-dependent oxygenases comprise a large family of enzymes that utilize an Fe(IV)-oxo intermediate to initiate diverse oxidative transformations with important biological roles. Here, four of the major types of Fe(II)/2OG-dependent reactions are detailed: hydroxylation, halogenation, ring formation, and desaturation. In addition, an atypical epimerization reaction is described. Studies identifying several key intermediates in catalysis are concisely summarized, and the proposed mechanisms are explained. In addition, a variety of other transformations catalyzed by selected family members are briefly described to further highlight the chemical versatility of these enzymes.

Analyzing the catalytic role of active site residues in the Fe-type nitrile hydratase from Comamonas testosteroni Ni1.

A strictly conserved active site arginine residue (αR157) and two histidine residues (αH80 and αH81) located near the active site of the Fe-type nitrile hydratase from Comamonas testosteroni Ni1 (CtNHase), were mutated. These mutant enzymes were examined for their ability to bind iron and hydrate acrylonitrile. For the αR157A mutant, the residual activity (k cat = 10 ± 2 s(-1)) accounts for less than 1% of the wild-type activity (k cat = 1100 ± 30 s(-1)) while the K m value is nearly unchanged at 205 ± 10 mM. On the other hand, mutation of the active site pocket αH80 and αH81 residues to alanine resulted in enzymes with k cat values of 220 ± 40 and 77 ± 13 s(-1), respectively, and K m values of 187 ± 11 and 179 ± 18 mM. The double mutant (αH80A/αH81A) was also prepared and provided an enzyme with a k cat value of 132 ± 3 s(-1) and a K m value of 213 ± 61 mM. These data indicate that all three residues are catalytically important, but not essential. X-ray crystal structures of the αH80A/αH81A, αH80W/αH81W, and αR157A mutant CtNHase enzymes were solved to 2.0, 2.8, and 2.5 Å resolutions, respectively. In each mutant enzyme, hydrogen-bonding interactions crucial for the catalytic function of the αCys(104)-SOH ligand are disrupted. Disruption of these hydrogen bonding interactions likely alters the nucleophilicity of the sulfenic acid oxygen and the Lewis acidity of the active site Fe(III) ion.

The active site sulfenic acid ligand in nitrile hydratases can function as a nucleophile.

Nitrile hydratase (NHase) catalyzes the hydration of nitriles to their corresponding commercially valuable amides at ambient temperatures and physiological pH. Several reaction mechanisms have been proposed for NHase enzymes; however, the source of the nucleophile remains a mystery. Boronic acids have been shown to be potent inhibitors of numerous hydrolytic enzymes due to the open shell of boron, which allows it to expand from a trigonal planar (sp(2)) form to a tetrahedral form (sp(3)). Therefore, we examined the inhibition of the Co-type NHase from Pseudonocardia thermophila JCM 3095 (PtNHase) by boronic acids via kinetics and X-ray crystallography. Both 1-butaneboronic acid (BuBA) and phenylboronic acid (PBA) function as potent competitive inhibitors of PtNHase. X-ray crystal structures for BuBA and PBA complexed to PtNHase were solved and refined at 1.5, 1.6, and 1.2 Å resolution. The resulting PtNHase-boronic acid complexes represent a "snapshot" of reaction intermediates and implicate the cysteine-sulfenic acid ligand as the catalytic nucleophile, a heretofore unknown role for the αCys(113)-OH sulfenic acid ligand. Based on these data, a new mechanism of action for the hydration of nitriles by NHase is presented.

The Fe-type nitrile hydratase from Comamonas testosteroni Ni1 does not require an activator accessory protein for expression in Escherichia coli.

We report herein the functional expression of an Fe-type nitrile hydratase (NHase) without the co-expression of an activator protein or the Escherichia coli chaperone proteins GroES/EL. Soluble protein was obtained when the α- and ß-subunit genes of the Fe-type NHase Comamonas testosteroni Ni1 (CtNHase) were synthesized with optimized E. coli codon usage and co-expressed. As a control, the Fe-type NHase from Rhodococcus equi TG328-2 (ReNHase) was expressed with (ReNHase(+Act)) and without (ReNHase(-Act)) its activator protein, establishing that expression of a fully functional, metallated ReNHase enzyme requires the co-expression of its activator protein, similar to all other Fe-type NHase enzymes reported to date, whereas the CtNHase does not. The X-ray crystal structure of CtNHase was determined to 2.4Å resolution revealing an αß heterodimer, similar to other Fe-type NHase enzymes, except for two important differences. First, two His residues reside in the CtNHase active site that are not observed in other Fe-type NHase enzymes and second, the active site Fe(III) ion resides at the bottom of a wide solvent exposed channel. The solvent exposed active site, along with the two active site histidine residues, are hypothesized to play a role in iron incorporation in the absence of an activator protein.