Non-heme dioxygenases: cellular sensors and regulators jelly rolled into one?Abdullah Ozer & Richard K Bruick

Members of the Fe(II)- and 2-oxoglutarate–dependent family of dioxygenases have long been known to oxidize several amino acids in various protein targets to facilitate protein folding. However, in recent years investigators have characterized several such hydroxylation modifications that serve a regulatory, rather than structural, purpose. Furthermore, the responsible enzymes seem to function directly as sensors of the cellular environment and metabolic state. For example, a cellular response pathway to low oxygen (hypoxia) is orchestrated through the actions of prolyl and asparaginyl hydroxylases that govern both the oxygen-dependent stability and transcriptional activity of the hypoxia-inducible transcription factor. Recently, a different subfamily of Fe(II)- and 2-oxoglutarate–dependent dioxygenases has been shown to carry out histone demethylation. The discovery of protein regulation via hydroxylation raises the possibility that other Fe(II)- and 2-oxoglutarate–dependent dioxygenases might also serve in a similar capacity.

Members of the Fe(II)- and 2-oxoglutarate–dependent family of dioxy-genases are found throughout biology to catalyze a number of oxida-tion reactions. Both the nature of the substrates and the consequences of oxidation vary immensely1. For example, deacetoxycephalosporin C synthase contributes to biosynthesis of cephalosporin antibiotics, AlkB repairs damaged DNA, and phytanoyl coenzyme A 2-hydroxylase is involved in the metabolism of a dietary fatty acid1. However, of particular recent interest are post-translational modifications of proteins catalyzed by members of this enzyme family.

Fe(II)- and 2-oxoglutarate–dependent dioxygenases are characterized by eight antiparallel β-strands that comprise the two β-sheets that con-stitute a jelly-roll motif1 (Fig. 1a). Unlike well-studied oxygenases such as cytochrome P450s, which use a heme-bound iron at their active sites, these dioxygenases directly bind Fe(II) through coordination of histi-dine, aspartate/glutamate, and histidine ligands (HXD/E…H motif) sandwiched between the β-sheets2. In addition to the primary protein substrate, 2-oxoglutarate is also a substrate for oxidation in the reaction and occupies two of the remaining iron coordination sites. A water ligand occupying the final iron coordination site must be dissociated before oxy-gen binding3. The incorporation of both oxygen atoms into organic prod-ucts further distinguishes these dioxygenases from P450 monooxygenases, which incorporate only one of the oxygen atoms into products—the other is reduced to water by reductants such as NADPH2 (ref. 4).

By analogy with other dioxygenases5,6, the post-translational modi-fication of proteins catalyzed by Fe(II)- and 2-oxoglutarate–dependent dioxygenases likely proceeds through a radical mechanism involving an iron-oxo intermediate7,8. As shown in Figure 1b, a quaternary complex composed of Fe(II), 2-oxoglutarate and substrate bound to the enzyme active site reacts with molecular oxygen (O2). An electron transferred

from Fe(II) generates a superoxide radical that attacks the C2 position of 2-oxoglutarate to form a covalent linkage between the Fe(IV) center and 2-oxoglutarate. Decarboxylation of the activated 2-oxoglutarate interme-diate produces succinate and CO2 with the concomitant formation of an Fe(IV)-oxo intermediate9,10. This Fe(IV)-oxo intermediate is then reduced upon abstraction of a hydrogen atom from the primary substrate, in the process generating a substrate-centered radical. Finally, carbon-oxygen bond formation yields the hydroxylated product while regenerating the active Fe(II) center. Overall, this reaction results in the transfer of one oxygen atom to the succinate byproduct and one to the protein substrate. The cofactor ascorbate is required for the full activity of this class of enzymes, presumably to maintain the ferrous state of iron1.

The fact that Fe(II)- and 2-oxoglutarate–dependent dioxygenases can modify proteins initially became evident from studies of structural pro-teins, as multiple proline residues within collagen and related proteins were found to be hydroxylated11. The hydroxyl group of 4-hydroxyproline is essential for proper assembly of collagen; it forms hydrogen bonds between the main chains of neighboring collagen polypeptides by bridg-ing water molecules12. The responsible collagen prolyl-4-hydroxylases (C-P4Hs) are obligate heterotetramers containing two copies each of their α and β subunits. The β subunit is a protein disulfide isomerase and does not contribute to the catalytic site for hydroxylation. Subsequent studies demonstrated that collagen contains additional hydroxylysine res-idues, which are also the product of Fe(II)- and 2-oxoglutarate–dependent dioxygenases13. In the intervening years many other amino acid side chains have been found to be hydroxylated by Fe(II)- and 2-oxoglutarate–dependent dioxygenases, including the side chains of asparagine14, aspar-tic acid15 and tryptophan16 (Fig. 1c). The consequences of many of these post-translational modifications remain unclear15,16. However, exciting new paradigms are emerging in which these modifications are engaged in signaling rather than structural roles, with the responsible dioxygenases serving as sensors of the cellular environment. An instructive example can be found in recent studies on a cellular pathway for sensing and responding to changes in oxygen availability.

Department of Biochemistry, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, Texas 75390-9038, USA. Correspondence should be addressed to R.K.B. (richard.bruick@utsouthwestern.edu).

Published online 14 February 2007; doi:10.1038/nchembio863

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The hypoxic response pathwayThe maintenance of cellular oxygen homeostasis is a critical process, as either excess (hyperoxia) or limiting (hypoxia) oxygen levels are harm-ful, resulting in excessive oxidative damage of biomolecules or cessa-tion of ATP production through oxidative phosphorylation, respectively. Because of oxygen’s vital importance, both local and systemic oxygen homeostasis is tightly controlled in metazoans through a number of pathways capable of responding to either chronic or acute changes in oxygen availability. One such pathway, which is conserved from worms to mammals and is ubiquitously expressed throughout each organism,

regulates an extensive array of genes in response to cellular hypoxia. Cellular hypoxia can arise under a variety of circumstances. These con-ditions can be physiological, such as the limi-tations to oxygen delivery by diffusion that are encountered during early embryogenesis, or from a shift to low-oxygen environments encountered at high altitudes17. Similarly, pathophysiological disease states can induce cellular hypoxia. For example, disruption of blood flow in myocardial or neuronal ischemia compromises oxygen delivery, whereas in solid tumors, cell growth that exceeds the local vas-culature’s capacity for blood delivery can result in intratumoral hypoxia17. The master regula-tor of this cellular hypoxic response pathway is a transcription factor called hypoxia-inducible factor (HIF) (Fig. 2).

When cells encounter a hypoxic environment, HIF is induced and in turn regulates a num-ber of gene products containing HIF response elements within their promoters. Most of the almost 100 HIF target genes characterized so far can be rationalized in regards to their ability to promote adaptation to hypoxic stress18. For example, the first characterized HIF-respon-sive gene encodes erythropoietin, which is produced primarily in the hypoxic kidney and liver to promote red blood cell maturation and enhance the oxygen-carrying capacity of the blood. HIF-dependent induction of vascular endothelial growth factor can further enhance oxygen delivery to hypoxic tissues by promoting new blood vessel growth. At the cellular level, induction of more than a dozen gene prod-ucts underlying glucose metabolism promotes anaerobic ATP production via glycolysis. Still other target genes mediate cellular survival and proliferation, extracellular matrix remodeling, and biosynthetic pathways.

HIF is composed of both an oxygen-sensitive HIF-α subunit and an oxygen-insensitive HIF-β subunit, which also doubles as the aryl-hydrocarbon receptor nuclear translocator (ARNT)19 (Fig. 2a). Both subunits feature basic helix-loop-helix (bHLH) and PER-ARNT-SIM (PAS) domains responsible for DNA binding and heterodimerization20. Three mamma-lian genes encode HIF-α subunits (HIF-1α, HIF-2α and HIF-3α), each of which is rapidly ubiquitinated when oxygen levels are high

(normoxia), thereby marking the subunit for proteasomal degrada-tion21–24. There are two HIF-α transactivation domains, the N-terminal transactivation domain (NTAD), which overlaps the region mediating HIF stability23, and an independent C-terminal transactivation domain (CTAD)25,26. Full HIF transcriptional activity requires recruitment of coactivators such as CREB-binding protein/p300 to the HIF-α subunit via the CTAD; such interactions are diminished under normoxia, how-ever14. Upon exposure to a hypoxic environment both HIF-α degrada-tion and CTAD coactivator recruitment blockage are bypassed, thereby allowing for HIF induction.

a b

c

NC

O

OH

NH2

O

C

NH

O

OH

4-Hydroxyproline β-Hydroxyasparagine

NH2C

NH

O

5-Hydroxylysine

OH

OHC

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O

OH

β-Hydroxyaspartic acid

O

C

NH

O

OH

3-Hydroxytryptophan

NH

NC

O

3-Hydroxyproline

HO

N

NH

–O

O

OO

Fe(II)

O O–

O

O

–O

Hisn

Hisn+x

Asp/Glun+2

N

NH

N

NH

–O

O

O

O

Fe(III)

O O–

O

O

–O

N

NH

–O

OO

O

Fe(IV)

O O–

O

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–O

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NH

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Fe(IV)

OO–

–OO

C

OO

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O

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Fe(III)

OO–

–OO

C

OO

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NH

–O

OFe(II)

OO–

–OO

C

OO

NH2

O

C

NH

O

N

NH

NH2

O

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NH

O

N

NH

NH2

O

C

NH

O

N

NH

NH2

O

C

NH

O

N

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NH2

O

C

NH

O

N

NH

NH2

O

C

NH

OOH H

Figure 1 Protein hydroxylation by Fe(II)- and 2-oxoglutarate–dependent dioxygenases. (a) Structural features of the Fe(II)- and 2-oxoglutarate–dependent dioxygenases. Shown here is the jelly-roll domain from the asparaginyl hydroxylase FIH-1 (Protein Data Bank (PDB) ID 1MZF), formed by two parallel sheets of four β-strands sandwiching the active site. A triad of histidine, aspartate and histidine residues coordinates binding of the catalytic ferrous iron (orange). A basic residue (lysine) contributes to the binding of the cosubstrate 2-oxoglutarate (green, with oxygen atoms in red). (b) Hydroxylated amino acid products of Fe(II)- and 2-oxoglutarate–dependent dioxygenases. (c) Proposed mechanism of Fe(II)- and 2-oxoglutarate–dependent dioxygenase-catalyzed reactions. In the example of asparaginyl hydroxylation catalyzed by FIH-1, the reaction proceeds through a radical mechanism involving an iron-oxo intermediate. Amino acid side chains of the enzyme are colored blue, the prime substrate asparagine is black, the 2-oxoglutarate cosubstrate is dark green, the succinnate and CO2 products are light green, and the oxygen atoms derived from molecular oxygen (O2) are red. The order of substrate binding may differ between members of this family3,117.

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Non-heme dioxygenases in the hypoxic response pathwayThough a number of hypotheses were proposed, the mechanism by which changes in oxygen availability are recognized by the cell to affect both HIF stability and activity remained elusive. The key to these mys-teries eventually came from the careful analysis of oxygen-dependent post-translational modifications in the HIF-α subunit in an unbiased manner by mass spectrometry. Strikingly, these data revealed HIF-α to be selectively hydroxylated under normoxia at two proline residues within the oxygen-dependent degradation domain (ODD)27–29, the region pre-viously shown to confer sensitivity to oxygen at the level of protein stabi-lity23. As shown in Figure 3a, modification of these residues is necessary for recognition of the HIF-α subunit by the product of the von Hippel-Lindau gene (pVHL), a component of the E3 ubiquitin ligase complex that targets HIF-α for proteasomal degradation30,31. Subsequent stud-ies revealed that HIF-α hydroxylation also extends to the CTAD, where hydroxylation of an asparagine residue under normoxia creates steric clashes responsible for abrogating the interaction with the coactivator p300 (ref. 14) (Fig. 3b).

These modifications provided a link between cellular oxygen concen-tration and HIF regulation, as under hypoxic conditions both prolyl and asparaginyl hydroxylation was diminished, which allowed for HIF-α sub-unit stabilization and coactivator recruitment. Drawing on the precedent of collagen hydroxylation by dioxygenases, HIF hydroxylation was also demonstrated to be dependent on iron and 2-oxoglutarate in vitro and in vivo32–34, thereby implicating this enzyme family in HIF regulation. Confirmation of this hypothesis came with the discovery of the respon-sible enzymes: a family of three mammalian HIF prolyl hydroxylases, alternatively named HPH-1/PHD3/EGLN3, HPH-2/PHD2/EGLN1 and HPH-3/PHD1/EGLN232–34, and a single HIF asparaginyl hydroxylase, named factor inhibiting HIF-1 (FIH-1)35,36.

As predicted, the HIF prolyl and asparaginyl hydroxylases are Fe(II)- and 2-oxoglutarate–dependent dioxygenases. As shown in the crystal structures determined for FIH-1 and the hydroxylase domain of HPH-2/PHD2/EGLN1 (Fig. 4a), both enzymes feature a characteristic β-jelly-roll domain topology orienting conserved residues that form the catalytic center37–40. Both active sites are composed of the distinctive iron-binding

Angiogenesis: VEGFErythropoiesis: EPOEnergy metabolism: GLUT1HIF regulation: HPH-1/PHD3/EGLN3 HPH-2/PHD2/EGLN1

Target genes

a

b

HIF-β

HIF-β HIF-α

p300coactivator

pVHL ubiquitinligase

complex

Pro564-OH

Asn803-OH

HRE

bHLH PAS A PAS B NTAD/ODD CTAD

Pro402 Pro564 Asn803

HIF-α

bHLH PAS A PAS B

HIF-β HIF-αpO2

2-OGFe(II)O2

Succinateα-ketoacids

HPH/PHD/EGLNFIH-1

Asc

ROS

?

?

Figure 2 Regulation of the mammalian hypoxia response pathway. (a) Domain features of the HIF subunits. (b) HIF regulation by Fe(II)- and 2-oxoglutarate–dependent dioxygenases. Under hypoxic conditions, the HIF heterodimer binds to HIF response elements (HREs) in the promoter regions of its target genes and recruits transcriptional coactivators such as p300 (CH1 domain shown in green) via the CTAD. Under normoxic conditions, the HIF-α subunit is hydroxylated by prolyl (HPH/PHD/EGLN) or asparaginyl (FIH-1) hydroxylases to promote recruitment of a ubiquitin ligase complex containing pVHL (yellow) or to block coactivator recruitment, respectively. Activities of the dioxygenases are regulated not only by the availability of oxygen, 2-oxoglutarate (2-OG) and iron, but are also sensitive to oxidative stress (ROS), metabolite concentrations (succinate, α-ketoacids and ascorbate (Asc)) and feedback loops. Representative structures were derived from PDB IDs 2A24, 1L3E, 2HBT, 1MZF and 1LM8. The structure of the MAX bHLH homodimer bound to DNA (PDB ID 1AN2) was used to represent HIF-α and HIF-β bHLH dimerization and DNA binding.

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HXD…H catalytic triad (Fig. 4b). 2-oxoglutarate is bound at the active site through interactions between the iron and the C1 carboxylate and C2 keto oxygens, in addition to a hydrogen bond between the C5 carboxylate group and a basic residue (Arg383 of HPH2 and Lys214 of FIH-1)37,38,40. Additional contacts between 2-oxoglutarate and FIH-1 are mediated by hydrogen bonds between the C5 carboxylate group of 2-oxoglutarate and the Tyr145 and Thr196 side chains, and through hydrophobic interac-tions with Leu188, Phe207 and Ile28137,38. Although the structure of the HIF prolyl hydroxylase domain does not include 2-oxoglutarate (it was cocrystallized with a competitive inhibitor), similar interactions between 2-oxoglutarate and the prolyl hydroxylases are anticipated40.

The HIF-α prolines marked for hydroxylation are found within the context of an LXXLAP motif, though substantial variation can be toler-ated in vitro41. In contrast to the short (~20 amino acid) model peptide substrates derived from the HIF-α ODD (Km ~ 7–8 µM)42, longer frag-ments encompassing the entire HIF-α ODD (amino acids 350–600) are much better substrates for the HIF prolyl hydroxylases (Km ~ 0.01–0.14 µM)43. This observation suggests that the enzyme-substrate interaction involves multiple contacts, some of which are far from the hydroxylation site. The narrow channel over the active site observed in the crystal struc-ture40 and the unstructured nature of the ODD44 are consistent with a model in which the target proline residue is inserted into the hydroxylase active site while the remainder of the ODD wraps around the enzyme in an extended conformation43,44. All three of the mammalian HIF prolyl hydroxylases recognize the C-terminal hydroxylation site (Pro564 of HIF-1α) with a higher affinity than the N-terminal hydroxylation site (Pro402 of HIF-1α), and one of the enzymes is unable to modify the latter site

at all42. Interestingly, hydroxylation at one site seems to promote hydroxylation at the other45, and this cooperativity between sites may con-tribute to the sharp transition in HIF induction observed at ~3% O2 in cell culture models46. Consistent with this observation, Pro564 hydroxylation precedes Pro402 hydroxy lation in vivo, and in the absence of Pro564, Pro402 hydroxylation is attenuated45.

In vitro, FIH-1 binds poorly to the HIF-α CTAD, with a Km ∼ 100 µM (ref. 47), though in vivo the FIH-1/HIF-α subunit interaction may be influenced by interactions with other factors48. Again, mutational analysis revealed a lack of conserved primary sequence elements underlying substrate selectivity49. The structure of FIH-1 bound to an HIF-1α CTAD peptide substrate shows two sites of interaction, one mediated by hydrogen bonds and the other predominantly comprised of hydrophobic interactions38. Residues at the former site adopt an extended conformation and surround the FIH-1 hydroxylation target (Asn803), which is buried into the active site of the enzyme38, whereas residues at the latter site remain in an α-helical structure when bound to either FIH-1 or p300 (refs. 38,50,51).

Beyond modification: HIF dioxygenases as sensorsThe idea that the enzymes responsible for the key regulatory modifications in the hypoxic response pathway consume oxygen as a sub-strate fueled speculation that these dioxygen-

ases serve as direct sensors of cellular oxygen availability, though such a hypothesis was not a foregone conclusion. For example, the collagen-modifying hydroxylases bind O2 rather well (C-P4H-1, Table 1) and are not limited by oxygen tensions known to induce HIF52. Km values for O2 measured with recombinant preparations of each of the four HIF hydroxylases range from 90 to 250 µM, with lower values reported when using longer (~250 amino acids versus ~20 amino acids) substrates42,43,47. These concentrations are comparable to concentrations of dissolved oxy-gen expected under hypoxic conditions, and they fit well with expecta-tions for a bona fide oxygen sensor42. A slightly greater decrease in oxygen levels is required to attenuate FIH-1 activity, which indicates the potential for differential HIF responses to oxygen, with the two enzymes tuned to sense different oxygen concentrations. Indeed, manipulation of FIH-1 expression at various O2 concentrations indicated that FIH-1’s enzy-matic activity persists under severe hypoxic conditions that otherwise inactivate the HIF prolyl hydroxylases53. Of course, it should be noted that the Km values of the four recombinant HIF hydroxylases prepared from different sources may not fully recapitulate the catalytic properties of these enzymes in vivo.

Despite the attractiveness of a model whereby the HIF dioxygenases serve as direct cellular oxygen sensors, there has remained some debate over the nature of oxygen sensing in this pathway. Alternative models have been proposed that implicate the mitochondria as the primary oxygen sensors, acting upstream of the HIF hydroxylases. Hypoxic sta-bilization of the HIF-α subunit is lost in cells treated with inhibitors of the mitochondrial electron transport chain54, though the underlying explanation for this observation remains unclear. It is possible that

Trp88

His115 Pro564

Tyr98

Trp117

Ser111

Asn803

Ala330

Lys334

Asp331Pro332

Arg335

Ile338

a

b

Figure 3 HIF-α hydroxylation mediates interactions with pVHL and p300. (a) Binding of a hydroxylated peptide derived from the HIF-1α ODD (magenta) to pVHL (PDB ID 1LM8)30,31. Key hydrogen bond contacts between pVHL and the hydroxylated proline are indicated on the right. (b) Binding of the HIF-1α CTAD (magenta) to the CH1 domain of p300 (PDB ID 1L3E)50,51. The β-carbon of the Asn803 residue, where hydroxylation occurs, is highlighted (magenta oval). The surface representations of pVHL and p300 are colored to reflect electrostatic charge (blue, positively charged amino acid side chains; red, negatively charged amino acid side chains).

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these effects arise when blockage of cellular oxygen consumption counteracts the non-physiological oxygen gradients encountered under most standard cell culture conditions, thereby raising the levels of oxygen available to the dioxygenases55,56. Conversely, advocates of the mitochondrial oxygen-sensing model pro-pose that under hypoxia endogenous hydroxy-lases are not limited for oxygen as a substrate. Instead the model holds that under hypoxic conditions the mitochondria generate a sig-nal, likely a subset of reactive oxygen species (ROS), that inactivates HIF hydroxylases by an as-yet-undetermined mechanism57–59.

Sensitivity of the HIF hydroxylases to oxi-dative stress has been implicated in other contexts as well. JunD, a member of the AP-1 transcription factor family, has recently been linked to HIF activation60. Because several JunD target genes encode antioxidant fac-tors, loss of JunD leads to accumulation of ROS. This increased cellular oxidative stress was found to be responsible for reduced HIF prolyl hydroxylase activity and subsequent HIF-α subunit stabilization and target gene induction60. The mechanism by which ROS might inhibit HIF hydroxylase activity remains undetermined. ROS effects could be mediated indirectly, through signaling pathways respon-sive to oxidative stress, or directly, through oxidation of redox-sensitive residues on the dioxygenases themselves. Another possibility is that ROS could directly oxidize the active site iron. Ascorbate is required for full activity of the HIF hydroxylases, likely as a result of its ability to act as a reducing agent to maintain the ferrous state of iron1. Interestingly, the physi-ological concentration of ascorbate (~25–50 µM)61 is well below Km values for ascorbate measured for the HIF hydroxylases42,47 (Table 1). Under these conditions, both dioxygenases may be prone to inhibition by oxidative stress, particularly under conditions that might further limit ascorbate concentrations62.

Cross talk between the mitochondria and the HIF hydroxylases is not limited to oxygen consumption or ROS-mediated processes. The activity of these dioxygenases also depends on the availability of the cosubstrate 2-oxoglutarate, a metabolic intermediate generated by the mitochondrial tricarboxylic acid cycle (TCA). Although the estimated cellular concentration of 2-oxoglutarate63 is near or well above the in vitro Km values of the HIF hydroxylases (Table 1), it is unclear what

the actual concentration of 2-oxoglutarate is in the nucleus and cyto-plasm, where these enzymes reside64,65. On the other hand, other TCA intermediates, such as succinate and fumarate, can inhibit hydroxy-lase activity by product inhibition or by competition for binding with 2-oxoglutarate66–68. It is tempting to speculate that relevant changes in the amounts of these intermediates may reflect the relative meta-bolic state of the cell, which is itself a function of the level of oxidative phosphorylation. Though data for such regulation under physiological conditions is still lacking, genetic mutations to TCA enzymes such as succinate dehydrogenase or fumarate hydratase68–70 have been observed in pheochromocytomas and renal cell carcinomas, in which they lead to accumulation of succinate or fumarate to concentrations sufficient to induce HIF even in the presence of O2.

Together these data argue for a privileged role for Fe(II)- and 2-oxoglutarate–dependent dioxygenases not only as regulatory modifiers of HIF stability and activity, but also as direct cellular sensors of oxygen, oxidative stress, and metabolites. These properties have spurred interest into other candidate dioxygenases with the hope that they too might have critical roles in the regulation of other biological pathways, and that they do so by operating in a sensing capacity. One exciting possibility has recently emerged with the discovery that a family of dioxygenases can post-translationally modify histones.

FIH-1 HPH-2/PHD2/EGLN1

Thr196His279

His199

Asp201

Phe207Leu188

Lys214

Tyr145

Ile281

His374 Asp315

His313Leu343

Ile327

Tyr329

Arg383

Val376

Tyr303

a

b

Figure 4 Structures of HIF prolyl (HPH-2/PHD2/EGLN1) and asparaginyl (FIH-1) hydroxylases. (a) Ribbon diagrams representing the structures of HIF hydroxylases37–40. The jelly-roll motifs are shown in red and the dimerization domain of FIH-1 is in yellow. (b) The active sites of the HIF hydroxylases feature residues required for Fe(II) (black sphere) and 2-oxoglutarate (gray) binding. The catalytic domain of HPH-2/PHD2/EGLN1 was crystallized with an inhibitor that occupies the 2-oxoglutarate binding site: [(4-hydroxy-8-iodoisoquinolin-3-yl)carbonyl]amino. Figures were generated from PDB IDs 1MZF and 2G19.

Table 1 Km values for Fe(II)- and 2-oxoglutarate–dependent dioxygenases

Km values (µM)

HPH-1 HPH-2 HPH-3 FIH-1 C-P4H-1

HIF-1α ODD 0.01–0.02a 0.14 ± 0.02a 0.07 ± 0.04a NA NA

HIF-1α CTAD NA NA NA 100 ± 5b NA

Oxygen 230c 100a; 250c 230c 90 ± 20b 40f

2-Oxoglutarate 60c <2e; 60c 55c 25 ± 3b 20f

Fe(II) 0.03 ± 0.002d 0.03 ± 0.004d 0.1 ± 0.04d 0.5 ± 0.2b 2f

Ascorbate 170c 180c 140c 260 ± 50b 300f

NA, not applicable. aSee ref. 43. bSee ref. 47. cSee ref. 42. dSee ref. 118. eSee ref. 119. fSee ref. 52.

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Non-heme dioxygenases as histone demethylasesIn order to accommodate a large genome within the nucleus, eukaryotic cells must compact their DNA. As this process necessarily sequesters the information encoded within the genome, cells have developed complex regulatory mechanisms to maintain the balance between DNA storage and accessibility. DNA condensation is initiated through the assembly of nucleosomes consisting of ~150 base pairs of DNA wrapped around a protein core composed of four core histone proteins (H2A, H2B, H3 and H4). Nucleosomes can in turn assemble into highly packed chro-matin fibers. The organization and subsequent accessibility of DNA in these structures is dynamic and is regulated at many levels. Of note, the N-terminal tails of the histone subunits are subject to extensive post-translational modifications, including (but not limited to) phosphoryla-tion, acetylation and methylation71. Each modification can affect others in ways that are not completely understood, and together they constitute a set of markers that reflect the local chromatin state, often referred to as the “histone code”71. Though some modifications may influence the strength of interactions between the DNA and histone proteins, these modifications frequently comprise binding sites for the recruitment of additional factors that further regulate DNA accessibility. Therefore the histone modifications must be dynamic—capable of being added or removed as necessary to accommodate processes such as DNA replica-tion and repair, and gene expression71.

Histone methylation has long been known to affect gene expression, though general rules relating specific methylation events to their effects on transcriptional activation or repression have proven to be more com-plicated than those of histone acetylation, which typically correlates with transcriptional activation72. In addition to the site of methylation, the extent and stereochemistry of methylation also affect the transcriptional outcome73. For many years investigators’ inability to identify demethyl-ation enzymes fueled speculation that methylation might irreversibly mark the local chromatin state74. Recently, lysine-specific demethylase 1 (LSD1) was shown to demethylate mono- and dimethylated lysines of histone H3 (ref. 75). LSD1 is an H3K4 (ref. 75) and H3K9 (ref. 76)

demethylase, abstracting hydride from dimethyllysine to form the corre-sponding imine while reducing an FAD cofactor75,77 (Scheme 1). FADH2 is then oxidized, thereby regenerating the FAD cofactor and releasing H2O2. Demethylation is completed by hydrolysis of the iminium to release formaldehyde75,77. Whether LSD1 is required for the final step is not clear, and the possibility of electron acceptors other than oxygen has also been raised77. Importantly, because the reaction catalyzed by LSD1 requires a protonated nitrogen on the lysine side chain, the enzyme can-not demethylate trimethyllysine75.

Interestingly, demethylation of an alkylated nitrogen by Fe(II)- and 2-oxoglutarate–dependent dioxygenases had also been demonstrated in a slightly different context. The Escherichia coli enzyme AlkB and its homologs repair DNA that has been damaged by alkylating agents by demethylating 1-methyladenine and 3-methylcytosine, and to a lesser extent 1-methylguanine and 3-methylthymine, to their original bases78. Iron-dependent oxidization of the methyl group is coupled to the oxida-tion of 2-oxoglutarate, and the oxidized methyl is then released as form-aldehyde78 (Scheme 1). By analogy, it was speculated that other members of the Fe(II)- and 2-oxoglutarate–dependent dioxygenase family might also demethylate proteins79. Further, because the demethylation reaction performed by these enzymes does not require a protonated nitrogen, this family was also expected to be capable of demethylating trimethylated lysines (Scheme 1).

This hypothesis was confirmed in the last year with the identifi-cation of several histone demethylases that operate via Fe(II)- and 2-oxoglutarate–dependent dioxygenation80–85 (Table 2). Though the proteins thus far shown to carry out this reaction contain numerous and varied domains, they each feature a jumonji C (JmjC) domain responsible for their demethylase activity and have been designated as jumonji histone demethylases (JHDM). Specific structural insights into the JmjC demethylase domain have come from the crystal structure of JHDM3A (ref. 86). Similar to FIH-1 and other Fe(II)- and 2-oxogluta-rate–dependent dioxygenases, the JmjC domain of JHDM3A adopts a jelly-roll fold containing an iron-binding HXD/E…H motif formed by

NH CH3

C

NH

O

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LSD1

N CH3C

NH

O

Trimethyllysine

CH3

2-OxoglutarateO2

SuccinateCO2

JHDMs

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1-Methyladenine

2-OxoglutarateO2

SuccinateCO2

AlkB

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N

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N

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Adenine

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O2H2O2

H2O

N

N

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N

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CH2OH

H2C O

H2C O

H2C O

N CH3C

NH

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CH2OH

CH3

H2NC

NH

O

Methyllysine

Dimethyllysine

C

NH

O

CH3CH2

CH3

NH CH3

CH3

Scheme 1 Demethylation reactions catalyzed by LSD1, AlkB and JHDMs. LSD1 can demethylate mono- and dimethyllysine via an FAD-mediated oxidation reaction. AlkB and members of the JHDM family are Fe(II)- and 2-oxoglutarate–dependent dioxygenases that demethylate 1-methyladenine and 3-methylcytosine, and mono-, di- and trimethyllysine residues, respectively. Carbon atoms of the methyl groups are highlighted in green.

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the His188, Glu190 and His276 residues (Fig. 5). Likewise, 2-oxoglutarate binding is stabilized by a basic residue (Lys206) in addition to hydrogen bond contacts with Tyr132 and Asn198 and hydrophobic interactions with Tyr177, Phe185 and Trp208 (ref. 86). Interestingly, the JHDM3A structure contains a rather unexpected feature: a zinc finger composed of residues from two different domains—Cys234 and His240 of the JmjC domain and Cys306 and Cys308 of a C-terminal domain86. Mutation of these residues destabilizes the protein in solution, which suggests that the zinc finger of JHDM3A serves a structural rather than regulatory function86.

Relatively little is yet known concerning the physiological roles of the demethylating dioxygenases. Because of their histone demethylase activ-ity, these regulatory enzymes are likely to have a broad impact on gene expression even though they may regulate a particular set of genes by specific recruitment with interacting proteins. For example, JHDM2A interacts with the androgen receptor in a hormone-dependent manner to enhance the expression of downstream target genes84, whereas JHDM3A associates with transcriptional corepressor complexes to downregulate gene expression87. Regardless, by analogy with the HIF hydroxylases, these histone demethylases might mediate changes in gene expression in direct response to changes in cellular levels of oxygen, ROS or metabolites.

DiscussionDespite the tremendous advances over the past five years, new insights into the roles of regulatory dioxygenases are emerging at a rapid pace. It is now clear that post-translational modification of proteins by Fe(II)- and 2-oxoglutarate–dependent dioxygenases is not simply limited to collagen-like substrates. Furthermore, the substrate and cofactor requirements of these enzymes have been exploited to confer sensitivity to changes in the cellular environment. Activity of the HIF hydroxylases is directly dependent on changes in cellular levels of oxygen, ROS and metabolic intermediates. The ‘sensing’ properties of these enzymes can be further influenced by interactions with other proteins48,88–91 or by changes in expression levels92. For example, the genes encoding two of the HIF pro-lyl hydroxylases are themselves targets of the HIF transcription factor,

and a drop in cellular oxygen availability increases hydroxylase expres-sion93,94. The resulting negative feedback loop provides for a more rapid degradation of HIF-α upon reoxygenation93,94. In addition, because these enzymes are frequently expressed at nonsaturating levels, such feedback provides a mechanism for resetting the normoxic oxygen set point93–95 by attenuating HIF-α concentrations under hypoxia93,95. Cells adapted to hypoxia for extended periods of time gradually lose HIF-α subunit stability, and they only induce HIF under severe hypoxia. Conversely, cells exposed to prolonged hyperoxia express low levels of HIF prolyl hydroxylases such that hydroxylation becomes limiting under otherwise normoxic conditions and HIF induction occurs at much higher oxygen levels. This phenomenon likely contributes to vasculature abnormalities observed in the eyes of premature newborns upon their removal from a hyperoxygenated incubator96. Together these multiple levels of regulation allow for dynamic responses to a range of cellular stresses that are tunable to the particular microenvironment of an individual cell. Though almost nothing is known regarding the regulation of the histone demethylases, it is tempting to speculate that they too mediate patterns of gene expression directly in response to environmental and metabolic cues throughout development and into adult life.

Already these dioxygenases are being exploited for potential therapeu-tic applications, as inhibitors of the HIF hydroxylases may be useful in treating anemias or in ameliorating ischemic damage34,40,97. Conversely, opportunities to enhance hydroxylase activity might prove useful in antagonizing HIF’s tumorigenic contributions61,98,99. Further insights will no doubt come from physiological studies of mouse knockout models100 or inactivating polymorphisms101. Interestingly, targeted disruption of the gene encoding HPH-2/PHD2/EGLN1 results in signifi-cant defects in heart development that are surprisingly not accompanied by an expected increase in HIF-α accumulation100. Such findings raise the possibility that the HIF hydroxylases may regulate other factors out-side of the HIF-dependent hypoxic response pathway. One candidate is the large subunit of RNA polymerase II, Rpb1 (ref. 102). Rpb1 is a VHL substrate that also contains a site for hydroxylation that is reminiscent of that of the modified prolines within the HIF-α ODD102.

Table 2 Known and putative human histone demethylases

Family Domain organization Examples Substrates References

JHDM1JHDM1A/FBXL11 H3K36me2, H3K36me 82

JHDM1B/FBXL10 H3K36me2 82

JHDM2

JHDM2A/JmjD1A/TSGA H3K9me2, H3K9me 84

JHDM2B ???

JHDM2C/TRIP8 ???

JHDM3

JHDM3A/JmjD2A H3K9me3, H3K9me2, H3K36me3, H3K36me2 80,81,83,85

JHDM3B/JmjD2B H3K9me3, H3K9me2, H3K36me2, H3K36me 80

JHDM3C/JmjD2C/GASC1 H3K9me3, H3K9me2, H3K36me3 80,83

JHDM3D/JmjD2D H3K9me3, H3K9me2 80,83

JmjC onlyJmjD4 ???

JmjD5 ???

Amine oxidase LSD1 H3K4me2, H3K4me, H3K9me2, H3K9me2 75,76

JmjC CXXC-ZF PHD FBOX LRR C2HC4-ZF JmjN Tudor SWIRM Amino oxidase

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HIF is just one of many transcription factors induced by hypoxia. For example, NF-κB has long been known to be regulated as a function of oxygen and oxidative stress. NF-κB is sequestered in the cytoplasm by IκB. Release of IκB from NF-κB is induced by phosphorylation of IκB by IκB kinases (IKKs), which are in turn activated by diverse stimuli. IKK-α and IKK-β each contain an LXXLAP motif and interact with HPH-3/PHD1/EGLN2 (ref. 103). Studies with pharmacological and small interfering RNA–based inhibitors of this HIF dioxygenase are consistent with a model in which HPH-3/PHD1/EGLN2 inhibits NF-κB activity by hydroxylation-dependent inactivation of IKK, and hypoxia in turn activates NF-κB103. The mechanism by which this putative modification inactivates the IKK remains unclear, as the protein is neither ubiquitinated nor degraded as a consequence of hydroxylation103. In addition, several ankyrin repeats found in proteins such as IκB and the precursor of the p50 subunit of NF-κB are substrates for the HIF asparaginyl hydroxylase, though again the physiological consequences of the modification have not yet been elucidated104. Nevertheless, these data suggest that the HIF hydroxylases might contribute to environmental and metabolic sensing and regulatory mechanisms in multiple cellular pathways.

Likewise, the physiological roles of the JmjC-containing Fe(II)- and 2-oxoglutarate–dependent dioxygenases need not be limited to the demethylation of lysine residues on histone subunits. In recent years several other proteins, including p53 (ref. 105), TAF10 (ref. 106) and cytochrome c (ref. 107), have been shown to be post-translationally methylated at a lysine residue. Reversal of this modification by these or related dioxygenases may contribute to the dynamic regulation of a number of factors. A similar modification found on histones and other proteins, including SMAD6 (ref. 108) and DNA polymerase β (ref. 109), is methylation of arginines. Though enzymes such as PAD4 can remove the methyl group by converting methylarginine to citrul-line110, no enzyme has yet been found to demethylate these residues back to arginine. From a chemical perspective, there is no reason why such Fe(II)- and 2-oxoglutarate–dependent dioxygenases would not be capable of reversing this modification as well.

Future investigations will almost certainly reveal an even richer biological niche for these and other members of the dioxygenase fam-ily15,111,112. Such candidate enzymes may also operate as sensors of oxygen, oxidative stress, metabolism, and perhaps still other stimuli as dictated by subtle differences in the enzyme active site that influence the chemical proper-ties of the bound iron. For instance, because the apparent Km values of HIF hydroxylases are well below the estimated cellular concentrations of bioavailable iron (~5–10 µM)113, these enzymes may not be sensitive to physiological fluctua-tions in iron availability except under condi-tions of extreme iron deprivation, such as those encountered upon incubation in the presence of iron chelators. Nevertheless, it has been sug-gested that other members of the Fe(II)- and 2-oxoglutarate–dependent dioxygenase fam-ily might have evolved to be more sensitive to changes in iron availability. Such factors have since been implicated as possible sensors and regulators of iron regulatory protein 2 (refs. 114,115), a key mediator of mammalian iron homeostasis116. Together, such avenues of investigation will no doubt extend the breadth of biology involving Fe(II)- and 2-oxoglutarate–

dependent dioxygenases, both as post-translational regulators of proteins and as direct sensors of the cellular milieu.

ACKNOWLEDGMENTSR.K.B. is the Michael L. Rosenberg Scholar in Medical Research and is supported by awards from the Burroughs Wellcome Fund and the Welch Foundation. We thank J. Ready for helpful comments.

COMPETING INTERESTS STATEMENTThe authors declare that they have no competing financial interests.

Published online at http://www.nature.com/naturechemicalbiologyReprints and permissions information is available online at http://npg.nature.com/reprintsandpermissions

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a b

Figure 5 Structure of the JmjC domain from the histone demethylase JHDM3A. (a) The overall structure of the catalytically active region of JHDM3A denoting iron (red sphere), zinc (blue sphere) and 2-oxoglutarate (gray, with oxygen atoms highlighted in red) locations. The JmjN and JmjC domains are colored in orange and green, respectively. (b) Substrate specificity determinants at the active site of JHDM3A. Residues within a flexible loop region (magenta) may discriminate between tri- and dimethyllysine substrate utilization among individual JHDM family members86.

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