Ex-Vivo 13C NMR Spectroscopy of Rodent Brain: TNF Restricts Neuronal Utilization of Astrocyte-Derived Metabolites.

Tumor necrosis factor (TNF) has well-established roles in neuroinflammatory disorders, but the effect of TNF on the biochemistry of brain cells remains poorly understood. Here, we microinjected TNF into the brain to study its impact on glial and neuronal metabolism (glycolysis, pentose phosphate pathway, citric acid cycle, pyruvate dehydrogenase, and pyruvate carboxylase pathways) using 13C NMR spectroscopy on brain extracts following intravenous [1,2-13C]-glucose (to probe glia and neuron metabolism), [2-13C]-acetate (probing astrocyte-specific metabolites), or [3-13C]-lactate. An increase in [4,5-13C]-glutamine and [2,3-13C]-lactate coupled with a decrease in [4,5-13C]-glutamate was observed in the [1,2-13C]-glucose-infused animals treated with TNF. As glutamine is produced from glutamate by astrocyte-specific glutamine synthetase the increase in [4,5-13C]-glutamine reflects increased production of glutamine by astrocytes. This was confirmed by infusion with astrocyte substrate [2-13C]-acetate. As lactate is metabolized in the brain to produce glutamate, the simultaneous increase in [2,3-13C]-lactate and decrease in [4,5-13C]-glutamate suggests decreased lactate utilization, which was confirmed using [3-13C]-lactate as a metabolic precursor. These results suggest that TNF rearranges the metabolic network, disrupting the energy supply chain perturbing the glutamine-glutamate shuttle between astrocytes and the neurons. These insights pave the way for developing astrocyte-targeted therapeutic strategies aimed at modulating effects of TNF to restore metabolic homeostasis in neuroinflammatory disorders.

Aromatic and antiaromatic ring currents in a molecular nanoring.

Aromatic and antiaromatic molecules-which have delocalized circuits of [4n + 2] or [4n] electrons, respectively-exhibit ring currents around their perimeters. The direction of the ring current in an aromatic molecule is such as to generate a magnetic field that opposes the external field inside the ring (a 'diatropic' current), while the ring current in an antiaromatic molecule flows in the reverse direction ('paratropic'). Similar persistent currents occur in metal or semiconductor rings, when the phase coherence of the electronic wavefunction is preserved around the ring. Persistent currents in non-molecular rings switch direction as a function of the magnetic flux passing through the ring, so that they can be changed from diatropic ('aromatic') to paratropic ('antiaromatic') simply by changing the external magnetic field. As in molecular systems, the direction of the persistent current also depends on the number of electrons. The relationship between ring currents in molecular and non-molecular rings is poorly understood, partly because they are studied in different size regimes: the largest aromatic molecules have diameters of about one nanometre, whereas persistent currents are observed in microfabricated rings with diameters of 20-1,000 nanometres. Understanding the connection between aromaticity and quantum-coherence effects in mesoscopic rings provides a motivation for investigating ring currents in molecules of an intermediate size. Here we show, using nuclear magnetic resonance spectroscopy and density functional theory, that a six-porphyrin nanoring template complex, with a diameter of 2.4 nanometres, is antiaromatic in its 4+ oxidation state (80 π electrons) and aromatic in its 6+ oxidation state (78 π electrons). The antiaromatic state has a huge paramagnetic susceptibility, despite having no unpaired electrons. This work demonstrates that a global ring current can be promoted in a macrocycle by adjusting its oxidation state to suppress the local ring currents of its components.The discovery of ring currents around a molecule with a circumference of 7.5 nanometres, at room temperature, shows that quantum coherence can persist in surprisingly large molecular frameworks.

Asymmetric Azidation under Hydrogen Bonding Phase-Transfer Catalysis: A Combined Experimental and Computational Study.

Asymmetric catalytic azidation has increased in importance to access enantioenriched nitrogen containing molecules, but methods that employ inexpensive sodium azide remain scarce. This encouraged us to undertake a detailed study on the application of hydrogen bonding phase-transfer catalysis (HB-PTC) to enantioselective azidation with sodium azide. So far, this phase-transfer manifold has been applied exclusively to insoluble metal alkali fluorides for carbon-fluorine bond formation. Herein, we disclose the asymmetric ring opening of meso aziridinium electrophiles derived from ß-chloroamines with sodium azide in the presence of a chiral bisurea catalyst. The structure of novel hydrogen bonded azide complexes was analyzed computationally, in the solid state by X-ray diffraction, and in solution phase by 1H and 14N/15N NMR spectroscopy. With N-isopropylated BINAM-derived bisurea, end-on binding of azide in a tripodal fashion to all three NH bonds is energetically favorable, an arrangement reminiscent of the corresponding dynamically more rigid trifurcated hydrogen-bonded fluoride complex. Computational analysis informs that the most stable transition state leading to the major enantiomer displays attack from the hydrogen-bonded end of the azide anion. All three H-bonds are retained in the transition state; however, as seen in asymmetric HB-PTC fluorination, the H-bond between the nucleophile and the monodentate urea lengthens most noticeably along the reaction coordinate. Kinetic studies corroborate with the turnover rate limiting event resulting in a chiral ion pair containing an aziridinium cation and a catalyst-bound azide anion, along with catalyst inhibition incurred by accumulation of NaCl. This study demonstrates that HB-PTC can serve as an activation mode for inorganic salts other than metal alkali fluorides for applications in asymmetric synthesis.

Metabolomics detects clinically silent neuroinflammatory lesions earlier than neurofilament-light chain in a focal multiple sclerosis animal model.

BACKGROUND: Despite widespread searches, there are currently no validated biofluid markers for the detection of subclinical neuroinflammation in multiple sclerosis (MS). The dynamic nature of human metabolism in response to changes in homeostasis, as measured by metabolomics, may allow early identification of clinically silent neuroinflammation. Using the delayed-type hypersensitivity (DTH) MS rat model, we investigated the serum and cerebrospinal fluid (CSF) metabolomics profiles and neurofilament-light chain (NfL) levels, as a putative marker of neuroaxonal damage, arising from focal, clinically silent neuroinflammatory brain lesions and their discriminatory abilities to distinguish DTH animals from controls. METHODS: 1H nuclear magnetic resonance (NMR) spectroscopy metabolomics and NfL measurements were performed on serum and CSF at days 12, 28 and 60 after DTH lesion initiation. Supervised multivariate analyses were used to determine metabolomics differences between DTH animals and controls. Immunohistochemistry was used to assess the extent of neuroinflammation and tissue damage. RESULTS: Serum and CSF metabolomics perturbations were detectable in DTH animals (vs. controls) at all time points, with the greatest change occurring at the earliest time point (day 12) when the neuroinflammatory response was most intense (mean predictive accuracy [SD]-serum: 80.6 [10.7]%, p < 0.0001; CSF: 69.3 [13.5]%, p < 0.0001). The top discriminatory metabolites at day 12 (serum: allantoin, cytidine; CSF: glutamine, glucose) were all reduced in DTH animals compared to controls, and correlated with histological markers of neuroinflammation, particularly astrogliosis (Pearson coefficient, r-allantoin: r = - 0.562, p = 0.004; glutamine: r = - 0.528, p = 0.008). Serum and CSF NfL levels did not distinguish DTH animals from controls at day 12, rather, significant differences were observed at day 28 (mean [SEM]-serum: 38.5 [4.8] vs. 17.4 [2.6] pg/mL, p = 0.002; CSF: 1312.0 [379.1] vs. 475.8 [74.7] pg/mL, p = 0.027). Neither serum nor CSF NfL levels correlated with markers of neuroinflammation; serum NfL did, however, correlate strongly with axonal loss (r = 0.641, p = 0.001), but CSF NfL did not (p = 0.137). CONCLUSIONS: While NfL levels were elevated later in the pathogenesis of the DTH lesion, serum and CSF metabolomics were able to detect early, clinically silent neuroinflammation and are likely to present sensitive biomarkers for the assessment of subclinical disease activity in patients.

Impact of Multiple Hydrogen Bonds with Fluoride on Catalysis: Insight from NMR Spectroscopy.

Hydrogen-bonding interactions have been explored in catalysis, enabling complex chemical reactions. Recently, enantioselective nucleophilic fluorination with metal alkali fluoride has been accomplished with BINAM-derived bisurea catalysts, presenting up to four NH hydrogen-bond donors (HBDs) for fluoride. These catalysts bring insoluble CsF and KF into solution, control fluoride nucleophilicity, and provide a chiral microenvironment for enantioselective fluoride delivery to the electrophile. These attributes encouraged a 1H/19F NMR study to gain information on hydrogen-bonding networks with fluoride in solution, as well as how these arrangements impact the efficiency of catalytic nucleophilic fluorination. Herein, NMR experiments enabled the determination of the number and magnitude of HB contacts to fluoride for thirteen bisurea catalysts. These data supplemented by diagnostic coupling constants 1hJNH···F- give insight into how multiple H bonds to fluoride influence reaction performance. In dichloromethane (DCM-d2), nonalkylated BINAM-derived bisurea catalyst engages two of its four NH groups in hydrogen bonding with fluoride, an arrangement that allows effective phase-transfer capability but low control over enantioselectivity for fluoride delivery. The more efficient N-alkylated BINAM-derived bisurea catalysts undergo urea isomerization upon fluoride binding and form dynamically rigid trifurcated hydrogen-bonded fluoride complexes that are structurally similar to their conformation in the solid state. Insight into how the countercation influences fluoride complexation is provided based on NMR data characterizing the species formed in DCM-d2 when reacting a bisurea catalyst with tetra-n-butylammonium fluoride (TBAF) or CsF. Structure-activity analysis reveals that the three hydrogen-bond contacts with fluoride are not equal in terms of their contribution to catalyst efficacy, suggesting that tuning individual electronic environment is a viable approach to control phase-transfer ability and enantioselectivity.

Reducing Agent-Mediated Nonenzymatic Conversion of 2-Oxoglutarate to Succinate: Implications for Oxygenase Assays.

l-Ascorbate (l-Asc) is often added to assays with isolated FeII - and 2-oxoglutarate (2OG)-dependent oxygenases to enhance activity. l-Asc is proposed to be important in catalysis by some 2OG oxygenases in vivo. We report observations on the nonenzymatic conversion of 2OG to succinate, which is mediated by hydrogen peroxide generated by the reaction of l-Asc and dioxygen. Slow nonenzymatic oxidation of 2OG to succinate occurs with some, but not all, other reducing agents commonly used in 2OG oxygenase assays. We intend these observations will help in the robust assignment of substrates and inhibitors for 2OG oxygenases.

Studies on the selectivity of proline hydroxylases reveal new substrates including bicycles.

Studies on the substrate selectivity of recombinant ferrous-iron- and 2-oxoglutarate-dependent proline hydroxylases (PHs) reveal that they can catalyse the production of dihydroxylated 5-, 6-, and 7-membered ring products, and can accept bicyclic substrates. Ring-substituted substrate analogues (such hydroxylated and fluorinated prolines) are accepted in some cases. The results highlight the considerable, as yet largely untapped, potential for amino acid hydroxylases and other 2OG oxygenases in biocatalysis.

19 F†NMR Monitoring of Reversible Protein Post-Translational Modifications: Class†D ß-Lactamase Carbamylation and Inhibition.

Bacterial production of ß-lactamases with carbapenemase activity is a global health threat. The active sites of class D carbapenemases such as OXA-48, which is of major clinical importance, uniquely contain a carbamylated lysine residue which is essential for catalysis. Although there is significant interest in characterizing this post-translational modification, and it is a promising inhibition target, protein carbamylation is challenging to monitor in solution. We report the use of 19 F NMR spectroscopy to monitor the carbamylation state of 19 F-labelled OXA-48. This method was used to investigate the interactions of OXA-48 with clinically used serine ß-lactamase inhibitors, including avibactam and vaborbactam. Crystallographic studies on 19 F-labelled OXA-48 provide a structural rationale for the sensitivity of the 19 F label to active site interactions. The overall results demonstrate the use of 19 F NMR to monitor reversible covalent post-translational modifications.

Global Aromaticity and Antiaromaticity in Porphyrin Nanoring Anions.

Doping, through oxidation or reduction, is often used to modify the properties of π-conjugated oligomers. In most cases, the resulting charge distribution is difficult to determine. If the oligomer is cyclic and doping establishes global aromaticity or antiaromaticity, then it is certain that the charge is fully delocalized over the entire perimeter of the ring. Herein we show that reduction of a six-porphyrin nanoring using decamethylcobaltocene results in global aromaticity (in the 6- state; [90 π]) and antiaromaticity (in the 4- state; [88 π]), consistent with the Hückel rules. Aromaticity is assigned by NMR spectroscopy and density-functional theory calculations.

Studies on the Substrate Selectivity of the Hypoxia-Inducible Factor Prolyl Hydroxylase†2 Catalytic Domain.

In animals, the response to chronic hypoxia is mediated by upregulation of the α,ß-heterodimeric hypoxia-inducible factors (HIFs). Levels of HIFα isoforms, but not HIFß, are regulated by their post-translational modification as catalysed by prolyl hydroxylase domain enzymes (PHDs). Different roles for the human HIF-1/2α isoforms and their two oxygen-dependent degradation domains (ODDs) are proposed. We report kinetic and NMR analyses of the ODD selectivity of the catalytic domain of wild-type PHD2 (which is conserved in nearly all animals) and clinically observed variants. Studies using Ala scanning and "hybrid" ODD peptides imply that the relatively rigid conformation of the (hydroxylated) proline plays an important role in ODD binding. They also reveal differential roles in binding for the residues on the N- and C-terminal sides of the substrate proline. The overall results indicate how the PHDs achieve selectivity for HIFα ODDs and might be of use in identifying substrate-selective PHD inhibitors.

Cyclobutanone Mimics of Intermediates in Metallo-ß-Lactamase Catalysis.

The most important resistance mechanism to ß-lactam antibiotics involves hydrolysis by two ß-lactamase categories: the nucleophilic serine and the metallo-ß-lactamases (SBLs and MBLs, respectively). Cyclobutanones are hydrolytically stable ß-lactam analogues with potential to inhibit both SBLs and MBLs. We describe solution and crystallographic studies on the interaction of a cyclobutanone penem analogue with the clinically important MBL SPM-1. NMR experiments using 19 F-labeled SPM-1 imply the cyclobutanone binds to SPM-1 with micromolar affinity. A crystal structure of the SPM-1:cyclobutanone complex reveals binding of the hydrated cyclobutanone through interactions with one of the zinc ions, stabilisation of the hydrate by hydrogen bonding to zinc-bound water, and hydrophobic contacts with aromatic residues. NMR analyses using a 13 C-labeled cyclobutanone support assignment of the bound species as the hydrated ketone. The results inform on how MBLs bind substrates and stabilize tetrahedral intermediates. They support further investigations on the use of transition-state and/or intermediate analogues as inhibitors of all ß-lactamase classes.

On the ozonolysis of unsaturated tosylhydrazones as a direct approach to diazocarbonyl compounds.

The scope and limitations are described of reacting unsaturated tosylhydrazones with O3 followed by Et3N for the generation of 1,4- and 1,5-diazocarbonyl systems. Tosylhydrazones, from tosylhydrazide condensation with readily available δ- and ε-unsaturated α-ketoesters, led in the former case to a 2-pyrazoline whereas the latter cases led to α-diazo-ε-ketoesters, although a terminal alkene produced a tetrahydropyridazinol. Using the ozonolysis-Et3N strategy, tosylhydrazones from cyclic enones give 2,5- and 2,6-diazoketones with aldehyde or ester functionality at the 1-position; the α-diazoaldehydes prefer the s-trans conformation, with a rotation barrier of 74 kJ mol-1 at 25 °C determined by NMR. This one-pot ozonolysis/Bamford-Stevens chemistry demonstrates both the tolerance of tosylhydrazones to ozone, and the subsequently added amine playing a dual role to directly transform the intermediate tosylhydrazone ozonides into products containing reactive diazo and ketone functionalities; such adducts are of particular value as precursors to cyclic carbonyl ylides for 1,3-dipolar cycloadditions.

Adenosine Monophosphate Binding Stabilizes the KTN Domain of the Shewanella denitrificans Kef Potassium Efflux System.

Ligand binding is one of the most fundamental properties of proteins. Ligand functions fall into three basic types: substrates, regulatory molecules, and cofactors essential to protein stability, reactivity, or enzyme-substrate complex formation. The regulation of potassium ion movement in bacteria is predominantly under the control of regulatory ligands that gate the relevant channels and transporters, which possess subunits or domains that contain Rossmann folds (RFs). Here we demonstrate that adenosine monophosphate (AMP) is bound to both RFs of the dimeric bacterial Kef potassium efflux system (Kef), where it plays a structural role. We conclude that AMP binds with high affinity, ensuring that the site is fully occupied at all times in the cell. Loss of the ability to bind AMP, we demonstrate, causes protein, and likely dimer, instability and consequent loss of function. Kef system function is regulated via the reversible binding of comparatively low-affinity glutathione-based ligands at the interface between the dimer subunits. We propose this interfacial binding site is itself stabilized, at least in part, by AMP binding.

Mechanistic Studies on a Cu-Catalyzed Asymmetric Allylic Alkylation with Cyclic Racemic Starting Materials.

Mechanistic studies on Cu-catalyzed asymmetric additions of alkylzirconocene nucleophiles to racemic allylic halide electrophiles were conducted using a combination of isotopic labeling, NMR spectroscopy, kinetic modeling, structure-activity relationships, and new reaction development. Kinetic and dynamic NMR spectroscopic studies provided insight into the oligomeric Cu-ligand complexes, which evolve during the course of the reaction to become faster and more highly enantioselective. The Cu-counterions play a role in both selecting different pathways and in racemizing the starting material via formation of an allyl iodide intermediate. We quantify the rate of Cu-catalyzed allyl iodide isomerization and identify a series of conditions under which the formation and racemization of the allyl iodide occurs. We developed reaction conditions where racemic allylic phosphates are suitable substrates using new phosphoramidite ligand D. D also allows highly enantioselective addition to racemic seven-membered-ring allyl chlorides for the first time. 1H and 2H NMR spectroscopy experiments on reactions using allylic phosphates showed the importance of allyl chloride intermediates, which form either by the action of TMSCl or from an adventitious chloride source. Overall these studies support a mechanism where complex oligomeric catalysts both racemize the starting material and select one enantiomer for a highly enantioselective reaction. It is anticipated that this work will enable extension of copper-catalyzed asymmetric reactions and provide understanding on how to develop dynamic kinetic asymmetric transformations more broadly.

Single-Acetylene Linked Porphyrin Nanorings.

The synthesis of ethyne-linked porphyrin nanorings has been achieved by template-directed Sonogashira coupling. The cyclic hexamer and octamer are predicted by density functional theory to adopt low symmetry conformations, due to dihedral twists between neighboring porphyrin units, but their symmetries are effectively D6h and D8h, respectively, in solution by 1H NMR. The fluorescence spectra indicate that the singlet excited states of these nanorings are highly delocalized.

Monitoring the Disassembly of Virus-like Particles by 19F-NMR.

Virus-like particles (VLPs) are stable protein cages derived from virus coats. They have been used extensively as biomolecular platforms, e.g., nanocarriers or vaccines, but a convenient in situ technique is lacking for tracking functional status. Here, we present a simple way to monitor disassembly of 19F-labeled VLPs derived from bacteriophage Qß by 19F NMR. Analysis of resonances, under a range of conditions, allowed determination not only of the particle as fully assembled but also as disassembled, as well as detection of a degraded state upon digestion by cells. This in turn allowed mutational redesign of disassembly and testing in both bacterial and mammalian systems as a strategy for the creation of putative, targeted-VLP delivery systems.

Terminally Truncated Isopenicillin N Synthase Generates a Dithioester Product: Evidence for a Thioaldehyde Intermediate during Catalysis and a New Mode of Reaction for Non-Heme Iron Oxidases.

Isopenicillin N synthase (IPNS) catalyses the four-electron oxidation of a tripeptide, l-δ-(α-aminoadipoyl)-l-cysteinyl-d-valine (ACV), to give isopenicillin N (IPN), the first-formed ß-lactam in penicillin and cephalosporin biosynthesis. IPNS catalysis is dependent upon an iron(II) cofactor and oxygen as a co-substrate. In the absence of substrate, the carbonyl oxygen of the side-chain amide of the penultimate residue, Gln330, co-ordinates to the active-site metal iron. Substrate binding ablates the interaction between Gln330 and the metal, triggering rearrangement of seven C-terminal residues, which move to take up a conformation that extends the final α-helix and encloses ACV in the active site. Mutagenesis studies are reported, which probe the role of the C-terminal and other aspects of the substrate binding pocket in IPNS. The hydrophobic nature of amino acid side-chains around the ACV binding pocket is important in catalysis. Deletion of seven C-terminal residues exposes the active site and leads to formation of a new type of thiol oxidation product. The isolated product is shown by LC-MS and NMR analyses to be the ene-thiol tautomer of a dithioester, made up from two molecules of ACV linked between the thiol sulfur of one tripeptide and the oxidised cysteinyl ß-carbon of the other. A mechanism for its formation is proposed, supported by an X-ray crystal structure, which shows the substrate ACV bound at the active site, its cysteinyl ß-carbon exposed to attack by a second molecule of substrate, adjacent. Formation of this product constitutes a new mode of reaction for IPNS and non-heme iron oxidases in general.

13C-Carbamylation as a mechanistic probe for the inhibition of class D ß-lactamases by avibactam and halide ions.

The class D (OXA) serine ß-lactamases are a major cause of resistance to ß-lactam antibiotics. The class D enzymes are unique amongst ß-lactamases because they have a carbamylated lysine that acts as a general acid/base in catalysis. Previous crystallographic studies led to the proposal that ß-lactamase inhibitor avibactam targets OXA enzymes in part by promoting decarbamylation. Similarly, halide ions are proposed to inhibit OXA enzymes via decarbamylation. NMR analyses, in which the carbamylated lysines of OXA-10, -23 and -48 were 13C-labelled, indicate that reaction with avibactam does not ablate lysine carbamylation in solution. While halide ions did not decarbamylate the 13C-labelled OXA enzymes in the absence of substrate or inhibitor, avibactam-treated OXA enzymes were susceptible to decarbamylation mediated by halide ions, suggesting halide ions may inhibit OXA enzymes by promoting decarbamylation of acyl-enzyme complex. Crystal structures of the OXA-10 avibactam complex were obtained with bromide, iodide, and sodium ions bound between Trp-154 and Lys-70. Structures were also obtained wherein bromide and iodide ions occupy the position expected for the 'hydrolytic water' molecule. In contrast with some solution studies, Lys-70 was decarbamylated in these structures. These results reveal clear differences between crystallographic and solution studies on the interaction of class D ß-lactamases with avibactam and halides, and demonstrate the utility of 13C-NMR for studying lysine carbamylation in solution.

Vernier templating and synthesis of a 12-porphyrin nano-ring.

Templates are widely used to arrange molecular components so they can be covalently linked into complex molecules that are not readily accessible by classical synthetic methods. Nature uses sophisticated templates such as the ribosome, whereas chemists use simple ions or small molecules. But as we tackle the synthesis of larger targets, we require larger templates-which themselves become synthetically challenging. Here we show that Vernier complexes can solve this problem: if the number of binding sites on the template, n(T), is not a multiple of the number of binding sites on the molecular building blocks, n(B), then small templates can direct the assembly of relatively large Vernier complexes where the number of binding sites in the product, n(P), is the lowest common multiple of n(B) and n(T) (refs 8, 9). We illustrate the value of this concept for the covalent synthesis of challenging targets by using a simple six-site template to direct the synthesis of a 12-porphyrin nano-ring with a diameter of 4.7 nm, thus establishing Vernier templating as a powerful new strategy for the synthesis of large monodisperse macromolecules.

Human oxygen sensing may have origins in prokaryotic elongation factor Tu prolyl-hydroxylation.

The roles of 2-oxoglutarate (2OG)-dependent prolyl-hydroxylases in eukaryotes include collagen stabilization, hypoxia sensing, and translational regulation. The hypoxia-inducible factor (HIF) sensing system is conserved in animals, but not in other organisms. However, bioinformatics imply that 2OG-dependent prolyl-hydroxylases (PHDs) homologous to those acting as sensing components for the HIF system in animals occur in prokaryotes. We report cellular, biochemical, and crystallographic analyses revealing that Pseudomonas prolyl-hydroxylase domain containing protein (PPHD) contain a 2OG oxygenase related in structure and function to the animal PHDs. A Pseudomonas aeruginosa PPHD knockout mutant displays impaired growth in the presence of iron chelators and increased production of the virulence factor pyocyanin. We identify elongation factor Tu (EF-Tu) as a PPHD substrate, which undergoes prolyl-4-hydroxylation on its switch I loop. A crystal structure of PPHD reveals striking similarity to human PHD2 and a Chlamydomonas reinhardtii prolyl-4-hydroxylase. A crystal structure of PPHD complexed with intact EF-Tu reveals that major conformational changes occur in both PPHD and EF-Tu, including a >20-Å movement of the EF-Tu switch I loop. Comparison of the PPHD structures with those of HIF and collagen PHDs reveals conservation in substrate recognition despite diverse biological roles and origins. The observed changes will be useful in designing new types of 2OG oxygenase inhibitors based on various conformational states, rather than active site iron chelators, which make up most reported 2OG oxygenase inhibitors. Structurally informed phylogenetic analyses suggest that the role of prolyl-hydroxylation in human hypoxia sensing has ancient origins.