Factor inhibiting HIF can catalyze two asparaginyl hydroxylations in VNVN motifs of ankyrin fold proteins.

The aspariginyl hydroxylase human factor inhibiting hypoxia-inducible factor (FIH) is an important regulator of the transcriptional activity of hypoxia-inducible factor. FIH also catalyzes the hydroxylation of asparaginyl and other residues in ankyrin repeat domain-containing proteins, including apoptosis stimulating of p53 protein (ASPP) family members. ASPP2 is reported to undergo a single FIH-catalyzed hydroxylation at Asn-986. We report biochemical and crystallographic evidence showing that FIH catalyzes the unprecedented post-translational hydroxylation of both asparaginyl residues in "VNVN" and related motifs of ankyrin repeat domains in ASPPs (i.e., ASPP1, ASPP2, and iASPP) and the related ASB11 and p18-INK4C proteins. Our biochemical results extend the substrate scope of FIH catalysis and may have implications for its biological roles, including in the hypoxic response and ASPP family function.

Biochemical and biophysical analyses of hypoxia sensing prolyl hydroxylases from Dictyostelium discoideum and Toxoplasma gondii.

In animals, the response to chronic hypoxia is mediated by prolyl hydroxylases (PHDs) that regulate the levels of hypoxia-inducible transcription factor α (HIFα). PHD homologues exist in other types of eukaryotes and prokaryotes where they act on non HIF substrates. To gain insight into the factors underlying different PHD substrates and properties, we carried out biochemical and biophysical studies on PHD homologues from the cellular slime mold, Dictyostelium discoideum, and the protozoan parasite, Toxoplasma gondii, both lacking HIF. The respective prolyl-hydroxylases (DdPhyA and TgPhyA) catalyze prolyl-hydroxylation of S-phase kinase-associated protein 1 (Skp1), a reaction enabling adaptation to different dioxygen availability. Assays with full-length Skp1 substrates reveal substantial differences in the kinetic properties of DdPhyA and TgPhyA, both with respect to each other and compared with human PHD2; consistent with cellular studies, TgPhyA is more active at low dioxygen concentrations than DdPhyA. TgSkp1 is a DdPhyA substrate and DdSkp1 is a TgPhyA substrate. No cross-reactivity was detected between DdPhyA/TgPhyA substrates and human PHD2. The human Skp1 E147P variant is a DdPhyA and TgPhyA substrate, suggesting some retention of ancestral interactions. Crystallographic analysis of DdPhyA enables comparisons with homologues from humans, Trichoplax adhaerens, and prokaryotes, informing on differences in mobile elements involved in substrate binding and catalysis. In DdPhyA, two mobile loops that enclose substrates in the PHDs are conserved, but the C-terminal helix of the PHDs is strikingly absent. The combined results support the proposal that PHD homologues have evolved kinetic and structural features suited to their specific sensing roles.

Synthesis of [4S-2 H]NADH, [4R-2 H]NADH, [4-2 H2 ]NADH and [4-2 H]NAD+ cofactors through heterogeneous biocatalysis in heavy water.

This practitioner protocol describes the synthesis of a family of deuterated nicotinamide cofactors: [4S-2 H]NADH, [4R-2 H]NADH, [4-2 H2 ]NADH and [4-2 H]NAD+ . The application of a recently developed H2 -driven heterogeneous biocatalyst enables the cofactors to be prepared with high (>90%) 2 H-incorporation with 2 H2 O as the only isotope source.

Kinetic Investigations of the Role of Factor Inhibiting Hypoxia-inducible Factor (FIH) as an Oxygen Sensor.

The hypoxia-inducible factor (HIF) hydroxylases regulate hypoxia sensing in animals. In humans, they comprise three prolyl hydroxylases (PHD1-3 or EGLN1-3) and factor inhibiting HIF (FIH). FIH is an asparaginyl hydroxylase catalyzing post-translational modification of HIF-α, resulting in reduction of HIF-mediated transcription. Like the PHDs, FIH is proposed to have a hypoxia-sensing role in cells, enabling responses to changes in cellular O2 availability. PHD2, the most important human PHD isoform, is proposed to be biochemically/kinetically suited as a hypoxia sensor due to its relatively high sensitivity to changes in O2 concentration and slow reaction with O2. To ascertain whether these parameters are conserved among the HIF hydroxylases, we compared the reactions of FIH and PHD2 with O2. Consistent with previous reports, we found lower Km(app)(O2) values for FIH than for PHD2 with all HIF-derived substrates. Under pre-steady-state conditions, the O2-initiated FIH reaction is significantly faster than that of PHD2. We then investigated the kinetics with respect to O2 of the FIH reaction with ankyrin repeat domain (ARD) substrates. FIH has lower Km(app)(O2) values for the tested ARDs than HIF-α substrates, and pre-steady-state O2-initiated reactions were faster with ARDs than with HIF-α substrates. The results correlate with cellular studies showing that FIH is active at lower O2 concentrations than the PHDs and suggest that competition between HIF-α and ARDs for FIH is likely to be biologically relevant, particularly in hypoxic conditions. The overall results are consistent with the proposal that the kinetic properties of individual oxygenases reflect their biological capacity to act as hypoxia sensors.

Oxygenase-catalyzed ribosome hydroxylation occurs in prokaryotes and humans.

The finding that oxygenase-catalyzed protein hydroxylation regulates animal transcription raises questions as to whether the translation machinery and prokaryotic proteins are analogously modified. Escherichia coli ycfD is a growth-regulating 2-oxoglutarate oxygenase catalyzing arginyl hydroxylation of the ribosomal protein Rpl16. Human ycfD homologs, Myc-induced nuclear antigen (MINA53) and NO66, are also linked to growth and catalyze histidyl hydroxylation of Rpl27a and Rpl8, respectively. This work reveals new therapeutic possibilities via oxygenase inhibition and by targeting modified over unmodified ribosomes.

A human protein hydroxylase that accepts D-residues.

Factor inhibiting hypoxia-inducible factor (FIH) is a 2-oxoglutarate-dependent protein hydroxylase that catalyses C3 hydroxylations of protein residues. We report FIH can accept (D)- and (L)-residues for hydroxylation. The substrate selectivity of FIH differs for (D) and (L) epimers, e.g., (D)- but not (L)-allylglycine, and conversely (L)- but not (D)-aspartate, undergo monohydroxylation, in the tested sequence context. The (L)-Leu-containing substrate undergoes FIH-catalysed monohydroxylation, whereas (D)-Leu unexpectedly undergoes dihydroxylation. Crystallographic, mass spectrometric, and DFT studies provide insights into the selectivity of FIH towards (L)- and (D)-residues. The results of this work expand the potential range of known substrates hydroxylated by isolated FIH and imply that it will be possible to generate FIH variants with altered selectivities.

Structural basis for oxygen degradation domain selectivity of the HIF prolyl hydroxylases.

The response to hypoxia in animals involves the expression of multiple genes regulated by the αß-hypoxia-inducible transcription factors (HIFs). The hypoxia-sensing mechanism involves oxygen limited hydroxylation of prolyl residues in the N- and C-terminal oxygen-dependent degradation domains (NODD and CODD) of HIFα isoforms, as catalysed by prolyl hydroxylases (PHD 1-3). Prolyl hydroxylation promotes binding of HIFα to the von Hippel-Lindau protein (VHL)-elongin B/C complex, thus signalling for proteosomal degradation of HIFα. We reveal that certain PHD2 variants linked to familial erythrocytosis and cancer are highly selective for CODD or NODD. Crystalline and solution state studies coupled to kinetic and cellular analyses reveal how wild-type and variant PHDs achieve ODD selectivity via different dynamic interactions involving loop and C-terminal regions. The results inform on how HIF target gene selectivity is achieved and will be of use in developing selective PHD inhibitors.

Inhibition of the HIF1α-p300 interaction by quinone- and indandione-mediated ejection of structural Zn(II).

Protein-protein interactions between the hypoxia inducible factor (HIF) and the transcriptional coactivators p300/CBP are potential cancer targets due to their role in the hypoxic response. A natural product based screen led to the identification of indandione and benzoquinone derivatives that reduce the tight interaction between a HIF-1α fragment and the CH1 domain of p300. The indandione derivatives were shown to fragment to give ninhydrin, which was identified as the active species. Both the naphthoquinones and ninhydrin were observed to induce Zn(II) ejection from p300 and the catalytic domain of the histone demethylase KDM4A. Together with previous reports on the effects of related compounds on HIF-1α and other systems, the results suggest that care should be taken in interpreting biological results obtained with highly electrophilic/thiol modifying compounds.

Reporter ligand NMR screening method for 2-oxoglutarate oxygenase inhibitors.

The human 2-oxoglutarate (2OG) dependent oxygenases belong to a family of structurally related enzymes that play important roles in many biological processes. We report that competition-based NMR methods, using 2OG as a reporter ligand, can be used for quantitative and site-specific screening of ligand binding to 2OG oxygenases. The method was demonstrated using hypoxia inducible factor hydroxylases and histone demethylases, and K(D) values were determined for inhibitors that compete with 2OG at the metal center. This technique is also useful as a screening or validation tool for inhibitor discovery, as exemplified by work with protein-directed dynamic combinatorial chemistry.

Asparaginyl beta-hydroxylation of proteins containing ankyrin repeat domains influences their stability and function.

Recent reports have provided evidence that the beta-hydroxylation of conserved asparaginyl residues in ankyrin repeat domain (ARD) proteins is a common posttranslational modification in animal cells. Here, nuclear magnetic resonance (NMR) and other biophysical techniques are used to study the effect of asparaginyl beta-hydroxylation on the structure and stability of 'consensus' ARD proteins. The NMR analyses support previous work suggesting that a single beta-hydroxylation of asparagine can stabilize the stereotypical ARD fold. A second asparaginyl beta-hydroxylation causes further stabilization. In combination with mutation studies, the biophysical analyses reveal that the stabilizing effect of beta-hydroxylation is, in part, mediated by a hydrogen bond between the asparaginyl beta-hydroxyl group and the side chain of a conserved aspartyl residue, two residues to the N-terminal side of the target asparagine. Removal of this hydrogen bond resulted in reduced stabilization by hydroxylation. Formation of the same hydrogen bond is also shown to be a factor in inhibiting binding of hydroxylated ARDs to factor-inhibiting hypoxia-inducible factor (FIH). The effects of hydroxylation appear to be predominantly localized to the target asparagine and proximal residues, at least in the consensus ARD protein. The results reveal that thermodynamic stability is a factor in determining whether a particular ARD protein is an FIH substrate; a consensus ARD protein with three ankyrin repeats is an FIH substrate, while more stable consensus ARD proteins, with four or five ankyrin repeats, are not. However, NMR studies reveal that the consensus protein with four ankyrin repeats is still able to bind to FIH, suggesting that FIH may interact in cells with natural ankyrin repeats without resulting hydroxylation. Overall, the work provides novel biophysical insights into the mechanism by which asparaginyl beta-hydroxylation stabilizes the ARD proteins and reduces their binding to FIH.

Evidence that two enzyme-derived histidine ligands are sufficient for iron binding and catalysis by factor inhibiting HIF (FIH).

A 2-His-1-carboxylate triad of iron binding residues is present in many non-heme iron oxygenases including the Fe(II) and 2-oxoglutarate (2OG)-dependent dioxygenases. Three variants (D201A, D201E, and D201G) of the iron binding Asp-201 residue of an asparaginyl hydroxylase, factor inhibiting HIF (FIH), were made and analyzed. FIH-D201A and FIH-D201E did not catalyze asparaginyl hydroxylation, but in the presence of a reducing agent, they displayed enhanced 2OG turnover when compared with wild-type FIH. Turnover of 2OG by FIH-D201A was significantly stimulated by the addition of HIF-1alpha(786-826) peptide. Like FIH-D201A and D201E, the D201G variant enhanced 2OG turnover but rather unexpectedly catalyzed asparaginyl hydroxylation. Crystal structures of the FIH-D201A and D201G variants in complex with Fe(II)/Zn(II), 2OG, and HIF-1alpha(786-826/788-806) implied that only two FIH-based residues (His-199 and His-279) are required for metal binding. The results indicate that variation of 2OG-dependent dioxygenase iron-ligating residues as a means of functional assignment should be treated with caution. The results are of mechanistic interest in the light of recent biochemical and structural analyses of non-heme iron and 2OG-dependent halogenases that are similar to the FIH-D201A/G variants in that they use only two His-residues to ligate iron.