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Auto-hydroxylation of FIH-1: an Fe(ii), alpha-ketoglutarate-dependent human hypoxia sensor.

ABSTRACT: HIF-asparaginyl hydroxylase (FIH-1) normally couples O(2)-activation to hydroxylation of Asn(803) on the alpha-subunit of the hypoxia-inducible factor (HIFalpha), a key step in pO(2) sensing; in the absence of HIFalpha, O(2)-activation becomes uncoupled, leading to self-hydroxylation at Trp(296) and a purple Fe(iii)-O-Trp chromophore-this alternative reactivity may affect human hypoxia sensing.

SUBMITTER: Chen YH

PROVIDER: S-EPMC2668816 | biostudies-literature | 2008 Oct

REPOSITORIES: biostudies-literature

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Auto-hydroxylation of FIH-1: an Fe(ii), alpha-ketoglutarate-dependent human hypoxia sensor.

Chen Yuan-Han YH Comeaux Lindsay M LM Eyles Stephen J SJ Knapp Michael J MJ

Chemical communications (Cambridge, England) 20080811 39

HIF-asparaginyl hydroxylase (FIH-1) normally couples O(2)-activation to hydroxylation of Asn(803) on the alpha-subunit of the hypoxia-inducible factor (HIFalpha), a key step in pO(2) sensing; in the absence of HIFalpha, O(2)-activation becomes uncoupled, leading to self-hydroxylation at Trp(296) and a purple Fe(iii)-O-Trp chromophore-this alternative reactivity may affect human hypoxia sensing. ...[more]

PMID: 18830487

Similar Datasets

Coordination changes and auto-hydroxylation of FIH-1: uncoupled O2-activation in a human hypoxia sensor.

Project description:Hypoxia sensing is the generic term for pO2-sensing in humans and other higher organisms. These cellular responses to pO2 are largely controlled by enzymes that belong to the Fe(II) alpha-ketoglutarate (alphaKG) dependent dioxygenase superfamily, including the human enzyme called the factor inhibiting HIF (FIH-1), which couples O2-activation to the hydroxylation of the hypoxia inducible factor alpha (HIFalpha). Uncoupled O2-activation by human FIH-1 was studied by exposing the resting form of FIH-1 (alphaKG + Fe)FIH-1, to air in the absence of HIFalpha. Uncoupling lead to two distinct enzyme oxidations, one a purple chromophore (lambda(max) = 583 nm) arising from enzyme auto-hydroxylation of Trp296, forming an Fe(III)-O-Trp296 chromophore [Y.-H. Chen, L.M. Comeaux, S.J. Eyles, M.J. Knapp, Chem. Commun. (2008), doi:10.1039/B809099H]; the other a yellow chromophore due to Fe(III) in the active site, which under some conditions also contained variable levels of an oxygenated surface residue (oxo)Met275. The kinetics of purple FIH-1 formation were independent of Fe(II) and alphaKG concentrations, however, product yield was saturable with increasing [alphaKG] and required excess Fe(II). Yellow FIH-1 was formed from (succinate+Fe)FIH-1, or by glycerol addition to (alphaKG+Fe)FIH-1, suggesting that glycerol could intercept the active oxidant from the FIH-1 active site and prevent hydroxylation. Both purple and yellow FIH-1 contained high-spin, rhombic Fe(III) centers, as shown by low temperature EPR. XAS indicated distorted octahedral Fe(III) geometries, with subtle differences in inner-shell ligands for yellow and purple FIH-1. EPR of Co(II)-substituted FIH-1 (alphaKG + Co)FIH-1, indicated a mixture of 5-coordinate and 6-coordinate enzyme forms, suggesting that resting FIH-1 can readily undergo uncoupled O2-activation by loss of an H2O ligand from the metal center.

| S-EPMC2649026 | biostudies-literature

Evidence that oxidative dephosphorylation by the nonheme Fe(II), α-ketoglutarate:UMP oxygenase occurs by stereospecific hydroxylation.

Project description:LipL and Cpr19 are nonheme, mononuclear Fe(II)-dependent, α-ketoglutarate (αKG):UMP oxygenases that catalyze the formation of CO2 , succinate, phosphate, and uridine-5'-aldehyde, the last of which is a biosynthetic precursor for several nucleoside antibiotics that inhibit bacterial translocase I (MraY). To better understand the chemistry underlying this unusual oxidative dephosphorylation and establish a mechanistic framework for LipL and Cpr19, we report herein the synthesis of two biochemical probes-[1',3',4',5',5'-2 H]UMP and the phosphonate derivative of UMP-and their activity with both enzymes. The results are consistent with a reaction coordinate that proceeds through the loss of one 2 H atom of [1',3',4',5',5'-2 H]UMP and stereospecific hydroxylation geminal to the phosphoester to form a cryptic intermediate, (5'R)-5'-hydroxy-UMP. Thus, these enzyme catalysts can additionally be assigned as UMP hydroxylase-phospholyases.

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Substrate Promotes Productive Gas Binding in the α-Ketoglutarate-Dependent Oxygenase FIH.

Project description:The Fe(2+)/α-ketoglutarate (αKG)-dependent oxygenases use molecular oxygen to conduct a wide variety of reactions with important biological implications, such as DNA base excision repair, histone demethylation, and the cellular hypoxia response. These enzymes follow a sequential mechanism in which O2 binds and reacts after the primary substrate binds, making those structural factors that promote productive O2 binding central to their chemistry. A large challenge in this field is to identify strategies that engender productive turnover. Factor inhibiting HIF (FIH) is a Fe(2+)/αKG-dependent oxygenase that forms part of the O2 sensing machinery in human cells by hydroxylating the C-terminal transactivation domain (CTAD) found within the HIF-1α protein. The structure of FIH was determined with the O2 analogue NO bound to Fe, offering the first direct insight into the gas binding geometry in this enzyme. Through a combination of density functional theory calculations, {FeNO}(7) electron paramagnetic resonance spectroscopy, and ultraviolet-visible absorption spectroscopy, we demonstrate that CTAD binding stimulates O2 reactivity by altering the orientation of the bound gas molecule. Although unliganded FIH binds NO with moderate affinity, the bound gas can adopt either of two orientations with similar stability; upon CTAD binding, NO adopts a single preferred orientation that is appropriate for supporting oxidative decarboxylation. Combined with other studies of related enzymes, our data suggest that substrate-induced reorientation of bound O2 is the mechanism utilized by the αKG oxygenases to tightly couple O2 activation to substrate hydroxylation.

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O<sub>2</sub> Activation by Nonheme Fe<sup>II</sup> α-Ketoglutarate-Dependent Enzyme Variants: Elucidating the Role of the Facial Triad Carboxylate in FIH.

Project description:FIH [factor inhibiting HIF (hypoxia inducible factor)] is an α-ketoglutarate (αKG)-dependent nonheme iron enzyme that catalyzes the hydroxylation of the C-terminal transactivation domain (CAD) asparagine residue in HIF-1α to regulate cellular oxygen levels. The role of the facial triad carboxylate ligand in O2 activation and catalysis was evaluated by replacing the Asp201 residue with Gly (D201G), Ala (D201A), and Glu (D201E). Magnetic circular dichroism (MCD) spectroscopy showed that the (FeII)FIH variants were all 6-coordinate (6C) and the αKG plus CAD bound FIH variants were all 5-coordinate (5C), mirroring the behavior of the wild-type ( wt) enzyme. When only αKG is bound, all FIH variants exhibited weaker FeII-OH2 bonds for the sixth ligand compared to wt, and for αKG-bound D201E this is either extremely weak or the site is 5C, demonstrating that the Asp201 residue plays an important role in the wt enzyme in ensuring that the (FeII/αKG)FIH site remains 6C. Variable-temperature, variable-field (VTVH) MCD spectroscopy showed that all of the αKG- and CAD-bound FIH variants, though 5C, have different ground-state geometric and electronic structures, which impair their oxygen activation rates. Comparison of O2 consumption to substrate hydroxylation kinetics revealed uncoupling between the two half reactions in the variants. Thus, the Asp201 residue also ensures fidelity between CAD substrate binding and oxygen activation, enabling tightly coupled turnover.

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Molecular insights into the unusually promiscuous and catalytically versatile Fe(II)/α-ketoglutarate-dependent oxygenase SptF.

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Cofactor Generation Cascade for ?-Ketoglutarate and Fe(II)-Dependent Dioxygenases.

Project description:Fe(II)- and ?-ketoglutarate dependent dioxygenases have emerged as important catalysts for the preparation of non-natural amino acids. The stoichiometric supply of the cosubstrate ?-ketoglutarate (?KG) is an important cost factor. A combination of the N-succinyl amino acid hydroxylase SadA with an l-glutamate oxidase (LGOX) allowed for coupling in situ production of ?KG to stereoselective ?KG-dependent dioxygenases in a one-pot/two-step cascade reaction. Both enzymes were used as immobilized enzymes and tested in a preparative scale setup under process-near conditions. Oxygen supply, enzyme, and substrate loading of the oxidation of glutamate were investigated under controlled reaction conditions on a small scale before upscaling to a 1 L stirred tank reactor. LGOX was applied with a substrate concentration of 73.6 g/L (339 mM) and reached a space-time yield of 14.2 g/L/h. Additionally, the enzyme was recycled up to 3 times. The hydroxylase SadA reached a space-time yield of 1.2 g/L/h at a product concentration of 9.3 g/L (40 mM). For both cascade reactions, the supply with oxygen was identified as a critical parameter. The results underline the robustness and suitability of ?-ketoglutarate dependent dioxygenases for application outside of living cells.

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The AlkB Family of Fe(II)/α-Ketoglutarate-dependent Dioxygenases: Repairing Nucleic Acid Alkylation Damage and Beyond.

Project description:The AlkB family of Fe(II)- and α-ketoglutarate-dependent dioxygenases is a class of ubiquitous direct reversal DNA repair enzymes that remove alkyl adducts from nucleobases by oxidative dealkylation. The prototypical and homonymous family member is an Escherichia coli "adaptive response" protein that protects the bacterial genome against alkylation damage. AlkB has a wide variety of substrates, including monoalkyl and exocyclic bridged adducts. Nine mammalian AlkB homologs exist (ALKBH1-8, FTO), but only a subset functions as DNA/RNA repair enzymes. This minireview presents an overview of the AlkB proteins including recent data on homologs, structural features, substrate specificities, and experimental strategies for studying DNA repair by AlkB family proteins.

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A Single-Turnover Kinetic Study of DNA Demethylation Catalyzed by Fe(II)/α-Ketoglutarate-Dependent Dioxygenase AlkB.

Project description:AlkB is a Fe(II)/α-ketoglutarate-dependent dioxygenase that repairs some alkylated bases of DNA and RNA in Escherichia coli. In the course of catalysis, oxidation of a co-substrate (α-ketoglutarate, αKG) leads to the formation of a highly reactive 'oxyferryl' enzyme-bound intermediate, Fe(IV) = O, ensuring hydroxylation of the alkyl nucleobase adducts. Previous studies have revealed that AlkB is a flexible protein and can adopt different conformations during interactions with cofactors and DNA. To assess the conformational dynamics of the enzyme in complex with single- or double-stranded DNA in real-time mode, we employed the stopped-flow fluorescence method. N1-Methyladenine (m1A) introduced into a sequence of 15-mer oligonucleotides was chosen as the specific damage. Single-turnover kinetics were monitored by means of intrinsic fluorescence of the protein's Trp residues, fluorescent base analogue 2-aminopurine (2aPu), and a dye-quencher pair (FAM/BHQ1). For all the fluorescent labels, the fluorescent traces showed several phases of consistent conformational changes, which were assigned to specific steps of the enzymatic process. These data offer an overall picture of the structural dynamics of AlkB and DNA during their interaction.

| S-EPMC6943663 | biostudies-literature

Electronic structure analysis of the oxygen-activation mechanism by Fe(II)- and α-ketoglutarate (αKG)-dependent dioxygenases.

Project description:α-Ketoglutarate (αKG)-dependent nonheme iron enzymes utilize a high-spin (HS) ferrous center to couple the activation of oxygen to the decarboxylation of the cosubstrate αKG to yield succinate and CO(2), and to generate a high-valent ferryl species that then acts as an oxidant to functionalize the target C-H bond. Herein a detailed analysis of the electronic-structure changes that occur in the oxygen activation by this enzyme was performed. The rate-limiting step, which is identical on the septet and quintet surfaces, is the nucleophilic attack of the distal O atom of the O(2) adduct on the carbonyl group in αKG through a bicyclic transition state ((5, 7) TS1). Due to the different electronic structures in (5, 7) TS1, the decay of (7)TS1 leads to a ferric oxyl species, which undergoes a rapid intersystem crossing to form the ferryl intermediate. By contrast, a HS ferrous center ligated by a peroxosuccinate is obtained on the quintet surface following (5)TS1. Thus, additional two single-electron transfer steps are required to afford the same Fe(IV)-oxo species. However, the triplet reaction channel is catalytically irrelevant. The biological role of αKG played in the oxygen-activation reaction is dual. The αKG LUMO (C=O π*) serves as an electron acceptor for the nucleophilic attack of the superoxide monoanion. On the other hand, the αKG HOMO (C1-C2 σ) provides the second and third electrons for the further reduction of the superoxide. In addition to density functional theory, high-level ab initio calculations have been used to calculate the accurate energies of the critical points on the alternative potential-energy surfaces. Overall, the results delivered by the ab initio calculations are largely parallel to those obtained with the B3LYP density functional, thus lending credence to our conclusions.

| S-EPMC3955955 | biostudies-literature

A Catharanthus roseus Fe(II)/α-ketoglutarate-dependent dioxygenase catalyzes a redox-neutral reaction responsible for vindolinine biosynthesis.

Project description:The Madagascar's periwinkle is the model plant for studies of plant specialized metabolism and monoterpenoid indole alkaloids (MIAs), and an important source for the anticancer medicine vinblastine. The elucidation of entire 28-step biosynthesis of vinblastine allowed further investigations for the formation of other remarkably complex bioactive MIAs. In this study, we describe the discovery and characterization of vindolinine synthase, a Fe(II)/α-ketoglutarate-dependent (Fe/2OG) dioxygenase, that diverts assembly of tabersonine to vinblastine toward the formation of three alternatively cyclized MIAs: 19S-vindolinine, 19R-vindolinine, and venalstonine. Vindolinine synthase catalyzes a highly unusual, redox-neutral reaction to form a radical from dehydrosecodine, which is further cyclized by hydrolase 2 to form the three MIA isomers. We further show the biosynthesis of vindolinine epimers from tabersonine using hydrolase 2 catalyzed reverse cycloaddition. While the occurrence of vindolinines is rare in nature, the more widely found venalstonine derivatives are likely formed from similar redox-neutral reactions by homologous Fe/2OG dioxygenases.

| S-EPMC9184523 | biostudies-literature

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