Dynamic Domain Links Substrate Binding and Catalysis in the Factor-Inhibiting-HIF-1.

The interplay between active-site chemistry and functionally relevant enzyme motions can provide useful insights into selective enzyme modulation. Modulation of the hypoxia-sensing function of factor-inhibiting-HIF-1 (FIH) enzyme is a potential therapeutic strategy in disease states such as ischemia and cancer. The hypoxia-sensing function of FIH relies in major part on the tight coupling of the first half of the catalytic mechanism which involves O activation and eventual succinate production to the second half which involves HIF-1α/CTAD substrate hydroxylation.

Measurement of kinetic isotope effects on peptide hydroxylation using MALDI-MS.

Primary kinetic isotope effects (KIEs) provide unique insight into enzymatic reactions, as they can reveal rate-limiting steps and detailed chemical mechanisms. HIF hydroxylases, part of a family of 2-oxoglutarate (2OG) oxygenases are central to the regulation of many crucial biological processes through O-sensing, but present a challenge to monitor due to the large size of the protein substrate and the similarity between native and hydroxylated substrate. MALDI-TOF MS is a convenient tool to measure peptide masses, which can also be used to measure the discontinuous kinetics of peptide hydroxylation for Factor Inhibiting HIF (FIH).

Kinetic Studies of the Hydrogen Atom Transfer in a Hypoxia-Sensing Enzyme, FIH-1: KIE and O Reactivity.

Cellular hypoxia plays a crucial role in tissue development and adaptation to pO. Central to cellular oxygen sensing is factor-inhibiting HIF-1α (FIH), an α-ketoglutarate (αKG)/non-heme iron(II)-dependent dioxygenase that hydroxylates a specific asparagine residue of hypoxia inducible factor-1α (HIF-1α). The high and rate-limiting decarboxylation step upon O activation are key features of the enzyme that classify it as an oxygen sensor and set it apart from other αKG/Fe(II)-dependent dioxygenases.

Protein Flexibility of the α-Ketoglutarate-Dependent Oxygenase Factor-Inhibiting HIF-1: Implications for Substrate Binding, Catalysis, and Regulation.

Protein dynamics are crucial for the mechanistically ordered enzymes to bind to their substrate in the correct sequence and perform catalysis. Factor-inhibiting HIF-1 (FIH) is a nonheme Fe(II) α-ketoglutarate-dependent oxygenase that is a key hypoxia (low ) sensor in humans. As these hypoxia-sensing enzymes follow a multistep chemical mechanism consuming α-ketoglutarate, a protein substrate that is hydroxylated, and O, understanding protein flexibility and the order of substrate binding may aid in the development of strategies for selective targeting.

Antioxidant capacity and anoxia tolerance in embryos.

Embryos of can tolerate extreme environmental stresses by entering into a state of metabolic and developmental arrest known as diapause. Oxidative stress is ubiquitous in aerobic organisms and the unique biology and ecology of likely results in frequent and repeated exposures to oxidative stress during development. The antioxidant capacity of was explored during development by measuring antioxidant capacity due to small molecules and several enzymatic antioxidant systems.

Chloride Supports O Activation in the D201G Facial Triad Variant of Factor-Inhibiting Hypoxia Inducible Factor, an α-Ketoglutarate Dependent Oxygenase.

α-Ketoglutarate (αKG) dependent oxygenases comprise a large superfamily of enzymes that activate O for varied reactions. While most of these enzymes contain a nonheme Fe bound by a His(Asp/Glu) facial triad, a small number of αKG-dependent halogenases require only the two His ligands to bind Fe and activate O. The enzyme "factor inhibiting HIF" (FIH) contains a HisAsp facial triad and selectively hydroxylates polypeptides; however, removal of the Asp ligand in the Asp201→Gly variant leads to a highly active enzyme, seemingly without a complete facial triad.

O Activation by Nonheme Fe α-Ketoglutarate-Dependent Enzyme Variants: Elucidating the Role of the Facial Triad Carboxylate in FIH.

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 O 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 (Fe)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.

Investigations on the role of a solvent tunnel in the α-ketoglutarate dependent oxygenase factor inhibiting HIF (FIH).

Non-heme Fe(II)/α-ketoglutarate (αKG)-dependent oxygenases catalyze a wide array of reactions through coupling oxidative decarboxylation of αKG to substrate oxygenation. This class of enzymes follows a sequential mechanism in which O reacts only after binding primary substrate, raising questions over how protein structure tailors molecular access to the Fe(II) cofactor. The enzyme "factor inhibiting hypoxia inducible factor" (FIH) senses pO in human cells by hydroxylating the C-terminal transactivation domain (CTAD), suggesting that structural elements limiting molecular access to the active site may limit the pO response.

The facial triad in the α-ketoglutarate dependent oxygenase FIH: A role for sterics in linking substrate binding to O activation.

The factor inhibiting hypoxia inducible factor-1α (FIH) is a nonheme Fe(II)/αKG oxygenase using a 2-His-1-Asp facial triad. FIH activates O via oxidative decarboxylation of α-ketoglutarate (αKG) to generate an enzyme-based oxidant which hydroxylates the Asn residue within the C-terminal transactivation domain (CTAD) of HIF-1α. Tight coupling of these two sequential reactions requires a structural linkage between the Fe(II) and the substrate binding site to ensure that O activation occurs after substrate binds.

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

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.

Increased Turnover at Limiting O2 Concentrations by the Thr(387) → Ala Variant of HIF-Prolyl Hydroxylase PHD2.

PHD2 is a 2-oxoglutarate, non-heme Fe(2+)-dependent oxygenase that senses O2 levels in human cells by hydroxylating two prolyl residues in the oxygen-dependent degradation domain (ODD) of HIF1α. Identifying the active site contacts that determine the rate of reaction at limiting O2 concentrations is crucial for understanding how this enzyme senses pO2 and may suggest methods for chemically altering hypoxia responses. A hydrogen bonding network extends from the Fe(II) cofactor through ordered waters to the Thr(387) residue in the second coordination sphere.

The rate-limiting step of O2 activation in the α-ketoglutarate oxygenase factor inhibiting hypoxia inducible factor.

Factor inhibiting HIF (FIH) is a cellular O-sensing enzyme, which hydroxylates the hypoxia inducible factor-1α. Previously reported inverse solvent kinetic isotope effects indicated that FIH limits its overall turnover through an O activation step (Hangasky J. A.

Substrate positioning by Gln(239) stimulates turnover in factor inhibiting HIF, an αKG-dependent hydroxylase.

Nonheme Fe(II)/αKG-dependent oxygenases catalyze diverse reactions, typically inserting an O atom from O2 into a C-H bond. Although the key to their catalytic cycle is the fact that binding and positioning of primary substrate precede O2 activation, the means by which substrate binding stimulates turnover is not well understood. Factor Inhibiting HIF (FIH) is a Fe(II)/αKG-dependent oxygenase that acts as a cellular oxygen sensor in humans by hydroxylating the target residue Asn(803), found in the C-terminal transactivation domain (CTAD) of hypoxia inducible factor-1.

Oxygen sensing strategies in mammals and bacteria.

The ability to sense and adapt to changes in pO2 is crucial for basic metabolism in most organisms, leading to elaborate pathways for sensing hypoxia (low pO2). This review focuses on the mechanisms utilized by mammals and bacteria to sense hypoxia. While responses to acute hypoxia in mammalian tissues lead to altered vascular tension, the molecular mechanism of signal transduction is not well understood.

First- and second-sphere contributions to Fe(II) site activation by cosubstrate binding in non-heme Fe enzymes.

Non-heme Fe(II) enzymes exhibit a general mechanistic strategy where binding all cosubstrates opens a coordination site on the Fe(II) for O2 activation. This study shows that strong-donor ligands, steric interactions with the substrate and second-sphere H-bonding to the facial triad carboxylate allow for five-coordinate site formation in this enzyme superfamily.

Substrate preference of the HIF-prolyl hydroxylase-2 (PHD2) and substrate-induced conformational change.

HIF prolyl-4-hydroxylase 2 (PHD2) is a non-heme Fe, 2-oxoglutarate (2OG) dependent dioxygenase that regulates the hypoxia inducible transcription factor (HIF) by hydroxylating two conserved prolyl residues in N-terminal oxygen degradation domain (NODD) and C-terminal oxygen degradation domain (CODD) of HIF-1α. Prior studies have suggested that the substrate preference of PHD2 arises from binding contacts with the β2β3 loop of PHD2. In this study we tested the substrate selectivity of PHD2 by kinetic competition assays, varied ionic strength, and global protein flexibility using amide H/D exchange (HDX).

Spectroscopic studies of the mononuclear non-heme Fe(II) enzyme FIH: second-sphere contributions to reactivity.

Factor inhibiting hypoxia-inducible factor (FIH) is an α-ketoglutarate (αKG)-dependent enzyme which catalyzes hydroxylation of residue Asn803 in the C-terminal transactivation domain (CAD) of hypoxia-inducible factor 1α (HIF-1α) and plays an important role in cellular oxygen sensing and hypoxic response. Circular dichroism (CD), magnetic circular dichroism (MCD), and variable-temperature, variable-field (VTVH) MCD spectroscopies are used to determine the geometric and electronic structures of FIH in its (Fe(II)), (Fe(II)/αKG), and (Fe(II)/αKG/CAD) forms. (Fe(II))FIH and (Fe(II)/αKG)FIH are found to be six-coordinate (6C), whereas (Fe(II)/αKG/CAD)FIH is found to be a 5C/6C mixture.

Imposing function down a (cupin)-barrel: secondary structure and metal stereochemistry in the αKG-dependent oxygenases.

The Fe(ii)/αketoglutarate (αKG) dependent oxygenases catalyze a diverse range of reactions significant in biological processes such as antibiotic biosynthesis, lipid metabolism, oxygen sensing, and DNA and RNA repair. Although functionally diverse, the eight-stranded β-barrel (cupin) and HX(D/E)XnH facial triad motifs are conserved in this super-family of enzymes. Crystal structure analysis of 25 αKG oxygenases reveals two stereoisomers of the Fe cofactor, Anti and Clock, which differ in the relative position of the exchangeable ligand position and the primary substrate.

Inverse solvent isotope effects arising from substrate triggering in the factor inhibiting hypoxia inducible factor.

Oxygen homeostasis plays a critical role in angiogenesis, erythropoiesis, and cell metabolism. Oxygen homeostasis is set by the hypoxia inducible factor-1α (HIF-1α) pathway, which is controlled by factor inhibiting HIF-1α (FIH). FIH is a non-heme Fe(II), α-ketoglutarate (αKG)-dependent dioxygenase that inhibits HIF-1α by hydroxylating the C-terminal transactivation domain (CTAD) of HIF-1α at HIF-Asn(803).

Inverse solvent isotope effects demonstrate slow aquo release from hypoxia inducible factor-prolyl hydroxylase (PHD2).

Prolyl hydroxylase domain 2 (PHD2) is deemed a primary oxygen sensor in humans, yet many details of its underlying mechanism are still not fully understood. (Fe(2+) + αKG)PHD2 is 6-coordinate, with a 2His/1Asp facial triad occupying three coordination sites, a bidentate α-ketoglutarate occupying two sites, and an aquo ligand in the final site. Turnover is thought to be initiated upon release of the aquo ligand, creating a site for O(2) to bind at the iron.

Screening chelating inhibitors of HIF-prolyl hydroxylase domain 2 (PHD2) and factor inhibiting HIF (FIH).

Two primary O(2)-sensors for humans are the HIF-hydroxylases, enzymes that hydroxylate specific residues of the hypoxia inducible factor-α (HIF). These enzymes are factor inhibiting HIF (FIH) and prolyl hydroxylase-2 (PHD2), each an α-ketoglutarate (αKG) dependent, non-heme Fe(II) dioxygenase. Although the two enzymes have similar active sites, FIH hydroxylates Asn(803) of HIF-1α while PHD2 hydroxylates Pro(402) and/or Pro(564) of HIF-1α.

The second coordination sphere of FIH controls hydroxylation.

The factor inhibiting HIF (FIH) is a proximate oxygen sensor for human cells, hydroxylating Asn(803) within the α-subunit of the hypoxia inducible factor (HIF). FIH is an α-ketoglutatrate (αKG)-dependent, non-heme Fe(II) dioxygenase, in which Fe(II) is coordinated by a (His(2)Asp) facial triad, αKG, and H(2)O. Hydrogen bonding among the facial triad, the HIF-Asn(803) side chain, and various second-sphere residues suggests a functional role for the second coordination sphere in tuning the chemistry of the Fe(II) center.

Uncoupled O2-activation in the human HIF-asparaginyl hydroxylase, FIH, does not produce reactive oxygen species.

The factor inhibiting HIF (FIH) is one of the primary oxygen sensors in human cells, controlling gene expression by hydroxylating the α-subunit of the hypoxia inducible transcription factor (HIF). As FIH is an alpha-ketoglutarate dependent non-heme iron dioxygenase, oxygen activation is thought to precede substrate hydroxylation. The coupling between oxygen activation and substrate hydroxylation was hypothesized to be very tight, in order for FIH to fulfill its function as a regulatory enzyme.

Optical explosives detection: from color changes to fluorescence turn-on.

The detection of chemical explosives is crucial for military and civilian safety. A confluence of chemistry and engineering continues to improve the sensitivity for several classes of explosives, and holds the promise of cheap and portable sensing. Optical and fluorescence-based sensors have been extensively researched for portable applications due to their sensitivity and portability.