Can a Mononuclear Iron(III)-Superoxo Active Site Catalyze the Decarboxylation of Dodecanoic Acid in UndA to Produce Biofuels?

Decarboxylation of fatty acids is an important reaction in cell metabolism, but also has potential in biotechnology for the biosynthesis of hydrocarbons as biofuels. The recently discovered nonheme iron decarboxylase UndA is involved in the biosynthesis of 1-undecene from dodecanoic acid and using X-ray crystallography was assigned to be a mononuclear iron species. However, the work was contradicted by spectroscopic studies that suggested UndA to be more likely a dinuclear iron system. To resolve this controversy we decided to pursue a computational study on the reaction mechanism of fatty acid decarboxylation by UndA using iron(III)-superoxo and diiron(IV)-dioxo models. We tested several models with different protonation states of active site residues. Overall, however, the calculations imply that mononuclear iron(III)-superoxo is a sluggish oxidant of hydrogen atom abstraction reactions in UndA and will not be able to activate fatty acid residues by decarboxylation at room temperature. By contrast, a diiron-dioxo complex reacts with much lower hydrogen atom abstraction barriers and hence is a more likely oxidant in UndA.

Does Substrate Positioning Affect the Selectivity and Reactivity in the Hectochlorin Biosynthesis Halogenase?

In this work we present the first computational study on the hectochlorin biosynthesis enzyme HctB, which is a unique three-domain halogenase that activates non-amino acid moieties tethered to an acyl-carrier, and as such may have biotechnological relevance beyond other halogenases. We use a combination of small cluster models and full enzyme structures calculated with quantum mechanics/molecular mechanics methods. Our work reveals that the reaction is initiated with a rate-determining hydrogen atom abstraction from substrate by an iron (IV)-oxo species, which creates an iron (III)-hydroxo intermediate. In a subsequent step the reaction can bifurcate to either halogenation or hydroxylation of substrate, but substrate binding and positioning drives the reaction to optimal substrate halogenation. Furthermore, several key residues in the protein have been identified for their involvement in charge-dipole interactions and induced electric field effects. In particular, two charged second coordination sphere amino acid residues (Glu223 and Arg245) appear to influence the charge density on the Cl ligand and push the mechanism toward halogenation. Our studies, therefore, conclude that nonheme iron halogenases have a chemical structure that induces an electric field on the active site that affects the halide and iron charge distributions and enable efficient halogenation. As such, HctB is intricately designed for a substrate halogenation and operates distinctly different from other nonheme iron halogenases.

The role of chloride in the mechanism of O(2) 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.

More than just a halogenase: modification of fatty acyl moieties by a trifunctional metal enzyme.

The highly selective oxidative halogenations by non-heme iron and α-ketoglutarate-dependent enzymes are key reactions in the biosynthesis of lipopeptides, and often bestow valuable bioactivity to the metabolites. Here we present the first biochemical characterization of a putative fatty acyl halogenase, HctB, which is found in the hectochlorin biosynthetic pathway of Lyngbya majuscula. Its unprecedented three-domain structure, which includes an acyl carrier protein domain, allows self-contained conversion of the covalently tethered hexanoyl substrate. Structural analysis of the native product by (13) C NMR reveals high regioselectivity but considerable catalytic promiscuity. This challenges the classification of HctB as a primary halogenase: along with the proposed dichlorination, HctB performs oxygenation and an unprecedented introduction of a vinyl-chloride moiety into the nonactivated carbon chain. The relaxed substrate specificity is discussed with reference to a molecular model of the enzyme-substrate complex. The results suggest that fatty acyl transformation at the metal center of HctB can bring about considerable structural diversity in the biosynthesis of lipopeptides.

Chiral hydroxylation at the mononuclear nonheme Fe(II) center of 4-(S) hydroxymandelate synthase--a structure-activity relationship analysis.

(S)-Hydroxymandelate synthase (Hms) is a nonheme Fe(II) dependent dioxygenase that catalyzes the oxidation of 4-hydroxyphenylpyruvate to (S)-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.

Structure and function of atypically coordinated enzymatic mononuclear non-heme-Fe(II) centers.

Mononuclear, non-heme-Fe(II) centers are key structures in O2 metabolism and catalyze an impressive variety of enzymatic reactions. While most are bound via two histidines and a carboxylate, some show a different organization. A short overview of atypically coordinated O2 dependent mononuclear-non-heme-Fe(II) centers is presented here Enzymes with 2-His, 3-His, 3-His-carboxylate and 4-His bound Fe(II) centers are discussed with a focus on their reactivity, metal ion promiscuity and recent progress in the elucidation of their enzymatic mechanisms. Observations concerning these and classically coordinated Fe(II) centers are used to understand the impact of the metal binding motif on catalysis.

Identification of human fumarylacetoacetate hydrolase domain-containing protein 1 (FAHD1) as a novel mitochondrial acylpyruvase.

The human fumarylacetoacetate hydrolase (FAH) domain-containing protein 1 (FAHD1) is part of the FAH protein superfamily, but its enzymatic function is unknown. In the quest for a putative enzymatic function of FAHD1, we found that FAHD1 exhibits acylpyruvase activity, demonstrated by the hydrolysis of acetylpyruvate and fumarylpyruvate in vitro, whereas several structurally related compounds were not hydrolyzed as efficiently. Conserved amino acids Asp-102 and Arg-106 of FAHD1 were found important for its catalytic activity, and Mg(2+) was required for maximal enzyme activity. FAHD1 was found expressed in all tested murine tissues, with highest expression in liver and kidney. FAHD1 was also found in several human cell lines, where it localized to mitochondria. In summary, the current work identified mammalian FAHD1 as a novel mitochondrial enzyme with acylpyruvate hydrolase activity.

Spectroscopic and computational studies of α-keto acid binding to Dke1: understanding the role of the facial triad and the reactivity of ß-diketones.

The O(2) activating mononuclear nonheme iron enzymes generally have a common facial triad (two histidine and one carboxylate (Asp or Glu) residue) ligating Fe(II) at the active site. Exceptions to this motif have recently been identified in nonheme enzymes, including a 3His triad in the diketone cleaving dioxygenase Dke1. This enzyme is used to explore the role of the facial triad in directing reactivity. A combination of spectroscopic studies (UV-vis absorption, MCD, and resonance Raman) and DFT calculations is used to define the nature of the binding of the α-keto acid, 4-hydroxyphenlpyruvate (HPP), to the active site in Dke1 and the origin of the atypical cleavage (C2-C3 instead of C1-C2) pattern exhibited by this enzyme in the reaction of α-keto acids with dioxygen. The reduced charge of the 3His triad induces α-keto acid binding as the enolate dianion, rather than the keto monoanion, found for α-keto acid binding to the 2His/1 carboxylate facial triad enzymes. The mechanistic insight from the reactivity of Dke1 with the α-keto acid substrate is then extended to understand the reaction mechanism of this enzyme with its native substrate, acac. This study defines a key role for the 2His/1 carboxylate facial triad in α-keto acid-dependent mononuclear nonheme iron enzymes in stabilizing the bound α-keto acid as a monoanion for its decarboxylation to provide the two additional electrons required for O(2) activation.

Exploring the catalytic potential of the 3-His mononuclear nonheme Fe(II) center: discovery and characterization of an unprecedented maltol cleavage activity.

Mononuclear nonheme iron enzymes (MNHEs) catalyze a range of very diverse reactions in O(2) metabolism, but they share a common principle active-site organization. To investigate a putative catalytic promiscuity of these enzymatic metal centers, we studied the reactivity of the 3-His ligated metal center of diketone cleaving enzyme (Dke1) toward non-native substrates, with a focus on alternative O(2) dependent reactions. From a screening approach, which aims at eliminating steric factors by including minimal substrate-substructures, three alternative, 'non-ß-dicarbonyl-cleavage' reactions are identified, among them an unprecedented oxygenation of maltol. Maltol cleavage is characterized by steady state and fast kinetic measurements and shows an O(2) concentration dependent rate determining step k(cat)/K(M)(O(2)) of 0.3mM(-1)s(-1) and a strict coupling of O(2) reduction and substrate oxidation. Furthermore, the catalytic potential of the 3-His metal center for O(2) dependent catechol ring-cleavage and phenylpyruvate oxidation (PP) is demonstrated.

The three-his triad in Dke1: comparisons to the classical facial triad.

The oxygen activating mononuclear non-heme ferrous enzymes catalyze a diverse range of chemistry yet typically maintain a common structural motif: two histidines and a carboxylate coordinating the iron center in a facial triad. A new Fe(II) coordinating triad has been observed in two enzymes, diketone-cleaving dioxygenase, Dke1, and cysteine dioxygenase (CDO), and is composed of three histidine residues. The effect of this three-His motif in Dke1 on the geometric and electronic structure of the Fe(II) center is explored via a combination of absorption, CD, MCD, and VTVH MCD spectroscopies and DFT calculations. This geometric and electronic structure of the three-His triad is compared to that of the classical (2-His-1-carboxylate) facial triad in the alpha-ketoglutarate (alphaKG)-dependent dioxygenases clavaminate synthase 2 (CS2) and hydroxyphenylpyruvate dioxygenase (HPPD). Comparison of the ligand fields at the Fe(II) shows little difference between the three-His and 2-His-1-carboxylate facial triad sites. Acetylacetone, the substrate for Dke1, will also bind to HPPD and is identified as a strong donor, similar to alphaKG. The major difference between the three-His and 2-His-1-carboxylate facial triad sites is in MLCT transitions observed for both types of triads and reflects their difference in charge. These studies provide insight into the effects of perturbation of the facial triad ligation of the non-heme ferrous enzymes on their geometric and electronic structure and their possible contributions to reactivity.

Kinetic and CD/MCD spectroscopic studies of the atypical, three-His-ligated, non-heme Fe2+ center in diketone dioxygenase: the role of hydrophilic outer shell residues in catalysis.

Diketone cleaving enzyme (Dke1) is a dioxygenase with an atypical, three-histidine-ligated, mononuclear non-heme Fe(2+) center. To assess the role in enzyme catalysis of the hydrophilic residues in the active site pocket, residues Glu98, Arg80, Tyr70, and Thr107 were subjected to mutational analysis. Steady state and pre-steady state kinetics indicated a role for Glu98 in promoting both substrate binding and O(2) reduction. Additionally, the Glu98 substitution eliminated the pH dependence of substrate binding (k(cat)(app)/K(M)(app)-pH profile) present in wild-type Dke1 (pK(a) = 6.3 +/- 0.4 and 8.4 +/- 0.4). MCD spectroscopy revealed that the Glu98 --> Gln mutation leads to the conversion of the six-coordinate (6C) resting Fe(2+) center present in the wild-type enzyme at pH 7.0 to a mixture of five-coordinate (5C) and 6C sites. The 6C geometry was restored with a pH shift to 9.5 which also resulted in ligand field (LF) energy splittings identical to that found for wild-type (WT) Dke1 at pH 9.5. In WT Dke1, these LF transitions are shifted up in energy by approximately 300 cm(-1) at pH 9.5 relative to pH 7.0. These data, combined with CD pH titrations which reveal a pK(a) of approximately 8.2 for resting WT Dke1 and the Glu98 --> Gln variant, indicate the deprotonation of a metal-ligated water. Together, the kinetic and spectroscopic data reveal a stabilizing effect of Glu98 on the 6C geometry of the metal center, priming it for substrate ligation. Arg80 and Tyr70 are shown to promote O(2) reduction, while Thr107 stabilizes the Fe(II) cofactor.

Functional characterization of an orphan cupin protein from Burkholderia xenovorans reveals a mononuclear nonheme Fe2+-dependent oxygenase that cleaves beta-diketones.

Cupins constitute a large and widespread superfamily of beta-barrel proteins in which a mononuclear metal site is both a conserved feature of the structure and a source of functional diversity. Metal-binding residues are contributed from two core motifs that provide the signature for the superfamily. On the basis of conservation of this two-motif structure, we have identified an ORF in the genome of Burkholderia xenovorans that encodes a novel cupin protein (Bxe_A2876) of unknown function. Recombinant Bxe_A2876, as isolated from Escherichia coli cell extract, was a homotetramer in solution, and showed mixed fractional occupancy of its 16.1 kDa subunit with metal ligands (0.06 copper; 0.11 iron; 0.17 zinc). Our quest for possible catalytic functions of Bxe_A2876 focused on Cu2+ and Fe2+ oxygenase activities known from related cupin enzymes. Fe2+ elicited enzymatic catalysis of O2-dependent conversion of various beta-diketone substrates via a nucleophilic mechanism of carbon-carbon bond cleavage. Data from X-ray absorption spectroscopy (XAS) support a five-coordinate or six-coordinate Fe2+ center where the metal is bound by three imidazole nitrogen atoms at 1.98 A. Results of structure modeling studies suggest that His60, His62 and His102 are the coordinating residues. In the 'best-fit' model, one or two oxygens from water and a carboxylate oxygen (presumably from Glu96) are further ligands of Fe2+ at estimated distances of 2.04 A and 2.08 A, respectively. The three-histidine Fe2+ site of Bxe_A2876 is compared to the mononuclear nonheme Fe2+ centers of the structurally related cysteine dioxygenase and acireductone dioxygenase, which also use a facial triad of histidines for binding of their metal cofactor but promote entirely different substrate transformations.

Why do cysteine dioxygenase enzymes contain a 3-His ligand motif rather than a 2His/1Asp motif like most nonheme dioxygenases?

Density functional theory calculations on the oxygen activation process in cysteine dioxygenase (CDO) and three active site mutants whereby one histidine group is replaced by a carboxylic acid group are reported. The calculations predict an oxygen activation mechanism that starts from an Fe(III)-O-O(*) complex that has close lying singlet, triplet, and quintet spin states. A subsequent spin state crossing to the quintet spin state surfaces leads to formation of a ring-structure whereby an O-S bond is formed. This weakens the central O-O bond, which is subsequently broken to give sulfoxide and an iron-oxo complex. The second oxygen atom is transferred to the substrate after a rotation of the sulfoxide group. A series of calculations were performed on cysteine dioxygenase mutants with a 2His/1Asp motif rather than a 3His motif. These calculations focused on the differences in catalytic and electronic properties of nonheme iron systems with a 3His ligand system versus a 2His/1Asp motif, such as taurine/alpha-ketoglutarate dioxygenase (TauD), and predict why CDO has a 3His ligand system while TauD and other dioxygenases share a 2His/1Asp motif. One mutant (H86D) had the ligand trans to the dioxygen group replaced by acetate, while in another set of calculations the ligand trans to the sulfur group of cysteinate was replaced by acetate (H88D). The calculations show that the ligands influence the spin state ordering of the dioxygen bound complexes considerably and in particular stabilize the quintet spin state more so that the oxygen activation step should encounter a lower energetic cost in the mutants as compared to WT. Despite this, the mutant structures require higher O-O bond breaking energies. Moreover, the mutants create more stable iron-oxo complexes than the WT, but the second oxygen atom transfer to the substrate is accomplished with much higher reaction barriers than the WT system. In particular, a ligand trans to the sulfur atom of cysteine that pushes electrons to the iron will weaken the Fe-S bond and lead to dissociation of this bond in an earlier step in the catalytic cycle than the WT structure. On the other hand, replacement of the ligand trans to the dioxygen moiety has minor effects on cysteinate binding but enhances the barriers for the second oxygen transfer process. These studies have given insight into why cysteine dioxygenase enzymes contain a 3His ligand motif rather than 2His/1Asp and show that the ligand system is essential for optimal dioxygenation activity of the substrate. In particular, CDO mutants with a 2His/1Asp motif may give sulfoxides as byproduct due to incomplete dioxygenation processes.

Biochemical characterization and mutational analysis of the mononuclear non-haem Fe2+ site in Dke1, a cupin-type dioxygenase from Acinetobacter johnsonii.

beta-diketone-cleaving enzyme Dke1 is a homotetrameric Fe2+-dependent dioxygenase from Acinetobacter johnsonii. The Dke1protomer adopts a single-domain beta-barrel fold characteristic of the cupin superfamily of proteins and features a mononuclear non-haem Fe2+ centre where a triad of histidine residues, His-62, His-64 and His-104, co-ordinate the catalytic metal. To provide structure-function relationships for the peculiar metal site of Dke1 in relation to the more widespread 2-His-1-Glu/Asp binding site for non-haem Fe2+,we replaced each histidine residue individually with glutamate and asparagine and compared binding of Fe2+ and four non-native catalytically inactive metals with purified apo-forms of wild-type and mutant enzymes. Results from anaerobic equilibrium microdialysis (Fe2+) and fluorescence titration (Fe2+, Cu2+, Ni2+, Mn2+ and Zn2+) experiments revealed the presence of two broadly specific metal-binding sites in native Dke1 that bind Fe2+ with a dissociation constant (Kd) of 5 microM (site I) and approximately 0.3 mM (site II). Each mutation, except for the substitution of asparagine for His-104, disrupted binding of Fe2+, but not that of the other bivalent metal ions, at site I,while leaving metal binding at site II largely unaffected. Dke1 mutants harbouring glutamate substitutions were completely inactive and not functionally complemented by external Fe2+.The Fe2+ catalytic centre activity (kcat) of mutants with asparagine substitution of His-62 and His-104 was decreased 140- and 220-fold respectively, compared with the kcat value of 8.5 s(-1) for the wild-type enzyme in the reaction with pentane-2,4-dione.The H64N mutant was not catalytically competent, except in the presence of external Fe2+ (1 mM) which elicited about 1/1000 of wild-type activity. Therefore co-ordination of Fe2+ by Dke1 requires an uncharged metallocentre, and three histidine ligands are needed for the assembly of a fully functional catalytic site. Oxidative inactivation of Dke1 was shown to involve conversion of enzyme-bound Fe2+ into Fe3+, which is then released from the metal centre.

Reaction coordinate analysis for beta-diketone cleavage by the non-heme Fe2+-dependent dioxygenase Dke1.

Acetylacetone dioxygenase from Acinetobacter johnsonii (Dke1) utilizes a non-heme Fe2+ cofactor to promote dioxygen-dependent conversion of 2,4-pentanedione (PD) into methylglyoxal and acetate. An oxidative carbon-carbon bond cleavage by Dke1 is triggered from a C-3 peroxidate intermediate that performs an intramolecular nucleophilic attack on the adjacent carbonyl group. But how does Dke1 bring about the initial reduction of dioxygen? To answer this question, we report here a reaction coordinate analysis for the part of the Dke1 catalytic cycle that involves O2 chemistry. A weak visible absorption band (epsilon approximately 0.2 mM(-1) cm(-1)) that is characteristic of an enzyme-bound Fe2+-beta-keto-enolate complex served as spectroscopic probe of substrate binding and internal catalytic steps. Transient and steady-state kinetic studies reveal that O2-dependent conversion of the chromogenic binary complex is rate-limiting for the overall reaction. Linear free-energy relationship analysis, in which apparent turnover numbers (k(app) cat) for enzymatic bond cleavage of a series of substituted beta-dicarbonyl substrates were correlated with calculated energies for the highest occupied molecular orbitals of the corresponding beta-keto-enolate structures, demonstrates unambiguously that k(app) cat is governed by the electron-donating ability of the substrate. The case of 2'-hydroxyacetophenone (2'HAP), a completely inactive beta-dicarbonyl analogue that has the enol double bond delocalized into the aromatic ring, indicates that dioxygen reduction and C-O bond formation cannot be decoupled and therefore take place in one single kinetic step.

Electronic substituent effects on the cleavage specificity of a non-heme Fe(2+)-dependent beta-diketone dioxygenase and their mechanistic implications.

Acinetobacter johnsonii acetylacetone dioxygenase (Dke1) is a non-heme Fe(II)-dependent dioxygenase that cleaves C-C bonds in various beta-dicarbonyl compounds capable of undergoing enolization to a cis-beta-keto enol structure. Results from 18O labeling experiments and quantitative structure-reactivity relationship analysis of electronic substituent effects on the substrate cleavage specificity of Dke1 are used to distinguish between two principle chemical mechanisms of reaction: one involving a 1,2-dioxetane intermediate and another proceeding via Criegee rearrangement. Oxygenative cleavage of asymmetrically substituted beta-dicarbonyl substrates occurs at the bond adjacent to the most electron-deficient carbonyl carbon. Replacement of the acetyl group in 1-phenyl-1,3-butanedione by a trifluoro-acetyl group leads to a complete reversal of cleavage frequency from 83% to only 8% fission of the bond next to the benzoyl moiety. The structure-activity correlation for Dke1 strongly suggests that enzymatic bond cleavage takes place via nucleophilic attack to generate a dioxetane, which then decomposes into the carboxylate and alpha-keto-aldehyde products.

Acetylacetone-cleaving enzyme Dke1: a novel C-C-bond-cleaving enzyme from Acinetobacter johnsonii.

The toxicity of acetylacetone has been demonstrated in various studies. Little is known, however, about metabolic pathways for its detoxification or mineralization. Data presented here describe for the first time the microbial degradation of acetylacetone and the characterization of a novel enzyme that initiates the metabolic pathway. From an Acinetobacter johnsonii strain that grew with acetylacetone as the sole carbon source, an inducible acetylacetone-cleaving enzyme was purified to homogeneity. The corresponding gene, coding for a 153 amino acid sequence that does not show any significant relationship to other known protein sequences, was cloned and overexpressed in Escherichia coli and gave high yields of active enzyme. The enzyme cleaves acetylacetone to equimolar amounts of methylglyoxal and acetate, consuming one equivalent of molecular oxygen. No exogenous cofactor is required, but Fe(2+) is bound to the active protein and essential for its catalytic activity. The enzyme has a high affinity for acetylacetone with a K (m) of 9.1 microM and a k(cat) of 8.5 s(-1). A metabolic pathway for acetylacetone degradation and the putative relationship of this novel enzyme to previously described dioxygenases are discussed.