Yes / 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.

Growing concerns of the negative effects on the environment and dependency of fossil fuelsare major driving forces for finding novel sustainable production pathways for plastic.Metabolic engineering has emerged as a powerful tool to enable microorganisms to producenon-native metabolites. The aim of this project was recombinant production of 4-hydroxybutyrate (4-HB) by expressing two enzymes in the model organism Escherichia coli.α-ketoglutarate decarboxylase (SucA) from Mycobacterium smegmatis followed by 4-hydroxybutyrate dehydrogenase (4-HBd) from Clostridium kluyveri was expressed inEscherichia coli. Results showed that the genes were successfully transformed and expressedin E. coli and after protein purification a concentration of 0.9 g/L SucA and 9.8 g/L 4-HBdwas achieved. Furthermore, some protein activity was detected by a coupled reaction withSucA and 4-HBd. When the enzymes got coupled together a change in NADH concentrationcould be detected spectrophotometrically. The enzymes were also tested for substratespecificity by using substrates with various carbon chain lengths and a decrease in NADHconcentration was seen. However, a decrease in the negative control for the experiments wasalso seen indicating a breakdown of NADH over time rather than consumption. Therefore, noconclusion could be drawn about the promiscuity of the enzymes. Lastly a single plasmidssystem was tested where both the genes were ligated on the same plasmid (pCDF duet) andexpressed successfully in E. coli Bl21DE3. / Ökad oro för miljön samt behovet av fossila resurser för produktion av plaster har gjort detnödvändigt att skapa nya och mer hållbara produktions vägar. Genetisk modifikation av olikaorganismer har utvecklats som ett starkt redskap för att få mikroorganismer att framställametaboliter som de normalt inte producerar. Målet med detta projekt var rekombinantproduktion av gamma hydroxibutansyra (4-HB) genom att uttrycka två enzym i modellorganismen Escherichia coli. Dessa enzym bestod av α-ketoglutarat dekarboxylas (SucA) frånMycobacterium smegmatis samt 4-hydroxybutyrate dehydrogenas (4-HBd) från Clostridiumkluyveri. Resultaten visade att proteinerna lyckades utryckas i E. coli med en koncentration av0,9 g/L SucA och 9,8 g/L 4-HBd som uppnåddes efter rening. Utöver detta detekterades ävenviss enzymaktivitet genom att kopplad enzymreaktion mellan 4-HBd och SucA och mätakonsumtionen av NADH spektrofotometriskt över tid. Enzymen testades även försubstratspecificitet genom att köra reaktionen med substrat med olika längd på kolkedjan. Dåkunde en minskning i NADH koncentrationen ses men det gjordes det även för de negativakontrollerna vilket indikerar nedbrytning av NADH och inte konsumtion av NADH. Ingaslutsatser angående enzymens substratspecificitet kunde därför dras. Det sista som gjordes varatt sätta in båda generna i ett en plasmidsystem där båda generna sattes in på samma plasmid(pCDF duet) och uttrycktes framgångsrikt i E. coli Bl21DE3.

Lors de ce travail, une étude comparative entre trois souches de C. glutamicum a été réalisée. Celles-ci sont C. glutamicum 2262, une souche surproductrice de glutamate suite à l’élévation de température du milieu de culture de 33 à 39°C, C. glutamicum 2262 NP un variant incapable d’excréter du glutamate dans ces mêmes conditions et C. glutamicum 2262 ∆pks13 un mutant dépourvu de bicouche mycolique externe. Un modèle métabolique original reprenant les différentes modifications physiologiques aboutissant à l’excrétion du glutamate au cours du procédé thermo-induit a été établi. La bicouche mycolique joue un rôle primordial puisque son absence affecte sévèrement la production du glutamate. Dans un premier temps, l’élévation de température serait ressentie au niveau de cette bicouche. Ce ressenti, visualisé par l’accumulation de protéines caractéristiques d’un stress thermique, est nécessaire pour que la bactérie soit en capacité de surproduire le glutamate. Par la suite, la production de glutamate est régulée au niveau de l’α-cétoglutarate déshydrogénase (ODH) grâce à la phosphoprotéine OdhI. Suite au changement de température, celle-ci est déphosphorylée ce qui lui permet d’interagir avec ODH et de provoquer l’inhibition de cette dernière. Ceci se traduit par la redirection sur flux carboné vers la synthèse du glutamate. Aucun de ces évènements n’est observé chez C. glutamicum 2262 ∆pks13. Par ailleurs, l’élévation de température induit une modification de la composition de l’enveloppe cellulaire qui semble intervenir dans le processus physiologique aboutissant à l’excrétion du glutamate puisque très peu de changements sont observés chez C. glutamicum 2262 NP / During this work, a comparative study between three strains of Corynebacterium glutamicum was carried out. These strains were C. glutamicum 2262 which overproduces glutamate after an increase in the culture temperature from 33 to 39°C, C. glutamicum 2262 NP which is unable to produce glutamate in the same culture conditions and C. glutamicum 2262 ∆pks13 devoid of outer corynomycolic acid bilayer. An original metabolic model describing the successive physiological modifications responsible for the glutamate excretion during the temperature-triggered process was established. The presence of the corynomycolic acid bilayer appeared to be necessary since its lack affected dramatically the glutamate production. The temperature increase would be first sensed at the level of the external corynomycolic acid layer. This sensing was visualised through the accumulation of thermal stress proteins. In C. glutamicum 2262 ∆pks13, the synthesis of these proteins was not induced. The glutamate production is regulated at the oxoglutarate dehydrogenase (ODH) level by the phosphoprotein OdhI. A consequence of the temperature increase was the dephosphorylation of this regulatory protein and its interaction with ODH, provoking its inhibition. The carbon flux was then reoriented toward the glutamate synthesis. In C. glutamicum 2262 ∆pks13, no dephosphorylation of OdhI and no change in the ODH activity were not determined. The thermal stress also induced a change in the composition of the corynomycolic acid layer which was correlated with the ability of C. glutamicum 2262 to overproduce glutamate. In C. glutamicum 2262 NP, the composition of the corynomycolic acid layer remained unchanged

2-0xoglutarate/Fe(II)-dependent dioxygenases (ZOG Oxygenases) are a relatively poorly characterised enzyme family that hydroxylate biological macromolecules to regulate a variety of essential cellular processes in mammals, including; chromatin remodeling, extra-cellular matrix formation and oxygen sensing. The work in this th esis focuses on a ZOG Oxygenase termed Myc-Induced Nuclear Antigen (MINAS3). This enzyme has been implicated in ribosome biogenesis and cell proliferation, and observed overexpressed in several tumour types, yet the identity afits substrate(s) and their role in cancer is unknown. The aims of the resea rch that has resulted in this thesis were to; (i) develop a cell model of MINAS3 enzyme activity, (ii) apply this model to study the role of MINAS3 activity in cell transformation and cancer, and (iii) discover novel cellular processes regulated by MINA53 activity. As such, I have created an isogenic cell model consisting of K-Ras-transformed MINAS3 knockout mouse embryonic fibroblasts (MEFs) reconstituted with either wildtype or enzyme-inactive MINAS3. Using this model I have shown that MINAS3 activity maintains normal levels of the large ribosomal subunit (60S), and suppresses anchorage-independent growth, autophagy and gene expression. These observations suggest the existence and involvement of one or more substrates. Indeed, proteomic and biochemical analyses in collaboration with the Schofield laboratory (Chemistry, Oxford) confirmed the identity of a MINA53 substrate, the 60S ribosomal protein Rp127a. Together we have shown that Rpl27a is abundantly hydroxylated, and that MINA53 is a histidinyJ hydroxylase; this represents the first discovery of a ribosomal oxygenase. The model developed here did not support a positive role for MINA53 in the transformation of MEFs. Rather it suggested that MINA53 can suppress transformation in some contexts, This prompted a wider investigation that demonstrated underexpression of MINA53 in several tumour types, and the presence of inactivating mutations in breast. ovarian and colon cancer. This thesis provides data supporting further research to understand the role of Rpl27a hydroxylation in the regulation of 60S biogenesis, autophagy and cancer. 2

Abstract Collagens and collagenous proteins undergo several post-translational modifications that are important for their structure and functions. Lysine hydroxylation produces hydroxylysines, which are important for collagen cross-link formation and provide attachment sites for galactose and glucosylgalactose. Glycosylated hydroxylysines are crucial for embryonic development and the assembly of certain collagen types. They may also facilitate interactions between collagen and adjacent molecules as well as control the diameter of collagen fibrils. Lysine hydroxylation is catalyzed by three lysyl hydroxylases (LH1, LH2 and LH3). In addition to lysyl hydroxylase activity, LH3 possesses collagen galactosyltransferase (GT) and glucosyltransferase (GGT) activities. In this study, polyclonal antibodies against the lysyl hydroxylase isoforms were produced for protein level studies to localize the expression and understand the functions of the different isoenzymes. The results indicated ubiquitous expression during embryonic development compared to the more restricted, cell and tissue specific expression patterns observed in adult mouse tissues. Differences were seen also in the alternative splicing of LH2 during embryogenesis and between tissue types. Analyses of the subcellular localization revealed that LH3 is also present in extracellular space. Tissue and cell specific differences were noted in the distribution of LH3 between cellular compartments. Substrate analysis suggested an additional and novel role for LH3 as an enzyme catalyzing lysine modifications of collagenous proteins in the extracellular space. The importance of LH1 and LH2 has been highlighted in Ehlers-Danlos type VI and Bruck syndromes, respectively. In this study, the lysyl hydroxylase 3 gene was linked to a heritable disorder for the first time. Urinary screening revealed a patient that lacked a glucosylgalactosyl derivative of a pyridinium cross-link. The GGT activity levels measured from the patient’s serum and lymphoblastoid cells were also reduced, which suggested a defect in the lysyl hydroxylase 3 gene. Genetic analyses revealed two mutations, one in each allele of LH3 in this compound heterozygous patient. Recombinant mutant proteins showed defects in lysyl hydroxylase and collagen glycosyltransferase activities, respectively. In conclusion, it was shown that a defect in LH3 catalyzed modifications leads to a novel disorder, which shares features with many other connective tissue disorders.

Oxygen dependent prolyl-4-hydroxylation of the alpha-subunit of the hypoxia inducible transcription factor (HIF-alpha) plays an essential role in the hypoxic response. Hydroxylation of proline residues in the N- or C-terminal oxygen dependent degradation domains (NODD or CODD) increases the affinity of HIF-alpha to the von Hippel-Lindau protein (pVHL) by approx. 1000 fold so signalling for HIF-alpha degradation. With limiting oxygen, HIF-alpha hydroxylation slows, it dimerises with HIF-beta and activates the transcription of a gene array. Prolyl-4-hydroxylation also stabilises the triple helix structure of collagen, the most abundant human protein. Both the collagen and the HIF prolyl hydroxylases (PHDs) are Fe(II) and 2-oxoglutarate (2OG) dependent oxygenases. Crystal structures of PHD2 in complex with CODD were determined in the current study. Together with biochemical analyses, the results demonstrate that catalysis involves a mobile region of PHD2 that encloses the hydroxylation site and stabilises the PHD2.Fe(II).2OG complex. When bound to PHD2 the pyrrolidine ring of the non-hydroxylated proline-residue adopts a C⁴-endo conformation. Evidence is provided that 4R-hydroxylation enables a stereoelectronic effect that changes the proline conformation to the C⁴-exo state, as observed when hydroxylated HIF-alpha is bound to pVHL and in collagen. The results help to rationalise NODD/CODD selectivity data for PHD isoforms and the effects of clinically observed mutations on PHD2 catalysis. Analyses on the interaction of nitric oxide with PHD2 are described and discussed with respect to regulation of the hypoxic response by nitric oxide.

This thesis explores roles of 2-oxoglutarate-dependent (2OG) oxygenases as interfaces that modulate steps in the flow of genetic information in cells in response to oxygen availability. Chapter 1 introduces mechanistic, biochemical and physiological aspects of major subfamilies of 2OG oxygenases, and their established regulatory roles in cells. In addition, structural and functional aspects of the ribosome and the translation process are discussed, with a focus on post-translational ribosome modifications. Chapter 2 investigates histone demethylases, which mediate chromatin-dependent regulation of gene expression and provides proof-of-concept for the rational, structure-guided design of small-molecules for selective inhibition of 2OG oxygenases with roles in cancer and inflammatory disease. Chapter 3 suggests regulatory roles for ten-eleven-translocation (TET)- catalysed DNA hydroxylation; calorimetric and thermal analyses reveal a duplex-stabilizing effect of the epigenetic 5-methylcytosine mark that is reversed upon conversion to 5- hydroxymethylcytosine (also termed the ‘sixth’ DNA base), raising the possibility that 2OG oxygenase catalysis might affect transcription via biophysical effects. Chapter 4 investigates fluoride release assays as a technology to enable medicinal chemistry studies on 2OG oxygenases with roles in fat mass regulation and obesity, cancer and inflammation; studies on the ALKBH5 enzyme show that it is a hypoxically upregulated 2OG oxygenase with a substrate preference distinct from previously characterized ALKBH enzymes. Chapter 5 identifies OGFOD1 as a 2OG-dependent ribosomal protein hydroxylase. OGFOD1 catalysis is conserved from yeast to humans. OGFOD1 catalyses formation of trans-3- hydroxy-L-proline in a highly conserved loop of ribosomal protein S23 proximal to the ribosomal decoding centre, possibly to modulate the interactions of eukaryotic ribosomes with tRNA, mRNA and translation factors in an oxygen-dependent manner. OGFOD1 is the functionally most well-conserved protein-modifying 2OG oxygenase; likewise, ribosomal protein S23 hydroxylation is the most well-conserved post-translational ribosome modification in eukaryotes. Some cell lines require OGFOD1 for proliferation, and scaffolds for OGFOD1- selective inhibitors are developed for use as potential antiproliferative agents and probes for cellular function. Chapter 6 shows the development of assays to investigate whether OGFOD1 catalysis affects ribosome assembly and function, including processivity, accuracy of initiation, elongation and termination, in yeast and mammalian cell lines. Chapter 7 concludes that ribosome hydroxylation might present an additional layer of regulatory complexity by which 2OG oxygenases could enable cells to respond to fluctuating oxygen levels.

Les furocoumarines sont des métabolites secondaires qui dérivent de la voie de biosynthèse des phénylpropanoïdes. Ces phytoalexines interviennent notamment dans les mécanismes de défense des plantes tels que la résistance aux bioagresseurs. Le déterminisme moléculaire de cette voie de biosynthèse est encore mal connu mais il a néanmoins été démontré depuis les années 1960 que les enzymes catalysant les différentes étapes de la voie la synthèse des furocoumarines appartenaient à différentes familles. Les travaux présentés dans ce document se sont focalisés sur deux familles d'enzymes : les dioxygenases oxoglutarate dépendantes et les cytochromes P450s. La première étape de la voie de biosynthèse des furocoumarines consiste en une ortho-hydroxylation du p-coumarate qui mène à la formation de l'umbelliférone. Basé sur des travaux récents sur Arabidopsis, six gènes codants pour des dioxygénases ont été isolés chez Ruta graveolens, Citrus limetta et Pastinaca sativa. Les protéines correspondantes présentent plus de 58% d'identité avec la Féruloyle 6' Hydroxylase (F6'H) d'A. thaliana. La caractérisation fonctionnelle de ces enzymes a été réalisée dans un système d'expression hétérologue procaryote. Sur les 6 enzymes, trois n'ont pu être exprimée efficacement, et deux présentent une activité F6'H similaire à celle décrite pour A. thaliana. La dernière enzyme dispose de caractéristiques nouvelles non décrites à ce jour. Elle est en mesure de réaliser l'hydroxylation du féruloyle coA et du p-coumaroyle coA. Ces études in vitro ont été complétées par une exploration des fonctions de la protéine dans la plante. Une analyse fine du profil d'expression du gène a permis de mettre en évidence une expression qui est en corrélation avec le niveau de production d'umbelliférone. La fonction de la protéine a également été validée par une analyse des produits formés dans des feuilles de Nicotiana benthamiana transformées transitoirement. Les cytochromes P450 catalysent 60% des réactions de la voie de biosynthèse de furocoumarines. Un travail de criblage fonctionnel de cytochromes P450 identifiés au préalable chez Ammi majus et Thapsia garganica a été entrepris. Les analyses de bio-informatiques et les modifications apportées au niveau du mode opératoire pour l'expression dans la levure ont permis d'émettre des hypothèses concernant le rôle de certains P450 candidats. Ces travaux exploratoires et préliminaires font supposer de nouvelles conjectures relatives à cette voie de biosynthèse / Furanocoumarins are secondary metabolites deriving from the phenylpropanoid biosynthetic pathway. These phytoalexins are especially involved in plant defense mechanisms against insects or phytopatogenous fungi and bacteria. The molecular control of this biosynthetic pathway is still poorly understood even though it has been demonstrated since the 1960s that enzymes catalyzing the different steps are belonging to different enzymatic families. The work presented here is focused on two enzyme families: oxoglutarate dependent dioxygenases and cytochrome P450s. The first step in the furanocoumarins biosynthetic pathway is the ortho-hydroxylation of p-coumarate, which leads to the formation of umbelliferone. Based on a recent work done on Arabidopsis, we isolated six genes encoding dioxygenases from Ruta graveolens, Citrus limetta and Pastinaca sativa. The corresponding proteins share more than 58% identity with the A. thaliana féruloyle 6' Hydroxylase (F6'H). The functional characterization of these enzymes was performed in a prokaryotic heterologous expression system. Of the six enzymes, three could not be functionnaly expressed and two exhibited a similar F6'H activity as described for A. thaliana. The last enzyme has new properties not described to date. It is able to achieve both hydroxylation of féruloyle CoA and p-coumaroyle CoA. These in vitro studies were completed by a functional exploration of the protein in planta. A detailed analysis of the gene expression pattern highlighted a link with the level of umbelliferone synthesis. The function of the protein was also confirmed by an analysis of the products formed in transiently transformed Nicotiana benthamiana leaves. Cytochrome P450s catalyze 60% of the reactions of the furanocoumarin biosynthetic pathway. Therefore, a functional screening of cytochrome P450 previously identified in Ammi majus and Thapsia garganica was undertaken. The bioinformatic analyses and the changes undertaken in the procedure for expression in yeast allowed drawing hypotheses on the function of some of these P450 candidates. These exploratory and preliminary experiments allowed suggesting new hypotheses about the biosynthetic pathway

Abstract 2-oxoglutarate-dependent dioxygenases (2-OGDDs) are an enzyme family that contains many enzymes that modify chromatin in extensive ways. These enzymes include several histone lysine demethylases (KDMs) and TET enzymes that convert methylated cytosine (5-mC) to 5-hydroxymethylcytosine (5-hmC) ultimately leading to DNA demethylation. Disturbed DNA and histone methylation are found in many cancers. However, the role of KDMs and TETs behind these oncogenic changes has so far not been fully investigated. This study focused on the role of these chromatin-modifying enzymes in cancers with special emphasis on enzyme kinetic studies. Cancers with inactivating fumarate hydratase (FH), succinate dehydrogenase (SDH) and isocitrate dehydrogenase (IDH) mutations accumulate fumarate, succinate and R-2-hydroxyglutarate, respectively. In this study we showed how these cancer-associated 2-oxoglutarate (2-OG) analogues can inhibit the TET enzymes and many of the KDMs leading to lower 5-hmC levels and increased H3K27 and H3K9 methylation on chromatin, respectively. We also characterized kinetic properties of acute myeloid leukaemia (AML)-associated TET2 mutants and found that their ability to bind 2-OG or iron was impaired leading to diminished catalytic activity. Tumours are often hypoxic due to inadequate vasculature and blood supply. The TET enzymes and KDMs require oxygen for the reactions they catalyse. We determined the oxygen affinity of TETs and many KDMs and found that a H3K27 demethylase KDM6A has a remarkably low affinity for oxygen indicating that it is inactivated in hypoxic tumours and tissues. H3K27 methylation was found to be increased in hypoxic cells and this blocked cell differentiation. Altogether, these studies shed light on the mechanisms behind the altered DNA and histone methylation found in several cancers with hypoxic conditions or FH, SDH and IDH mutations. Altered DNA and histone methylation has previously been associated with progression of cancer, such as epithelial-to-mesenchymal transition (EMT). We now linked catalytic inhibition of 2-OGDDs to disturbed DNA and histone methylation that can account for altered cell differentiation, EMT and increased aggressiveness and invasiveness of cancers. / Tiivistelmä 2-oksoglutaraatista riippuvaiset dioksygenaasit ovat entsyymiperhe, johon kuuluu useita entsyymejä, jotka muokkaavat kromatiinin epigeneettisiä merkkejä monin tavoin. Näitä entsyymejä ovat mm. DNA:n demetylaatioon vaikuttavat TET-entsyymit sekä useat histonidemetylaasit. Vaikka muutoksia DNA:n ja histonien metylaatiotasoissa on havaittu useissa syövissä, ei näiden entsyymien roolia muutosten taustalla ole vielä tutkittu. Tämä tutkimus kohdistui näiden epigenetiikkaan vaikuttavien entsyymien roolin ymmärtämiseen syövissä keskittyen erityisesti kyseisten entsyymien kinetiikkaan. Useissa syövissä on havaittu fumaraattihydrataasin, sukkinaattidehydrogenaasin ja isositraattidehydrogenaasien aktiivisuuteen vaikuttavia mutaatioita, jotka johtavat fumaraatin, sukkinaatin ja R-2-hydroksiglutaraatin kertymiseen syöpäsoluihin. Tässä tutkimuksessa osoitimme, kuinka nämä karsinogeeniset 2-oksoglutaraattianalogit voivat inhiboida TET-entsyymejä ja histonidemetylaaseja, mikä alentaa 5-hydroksimetyylisytosiinitasoja ja lisää histonien metylaatiota. Näytämme myös, kuinka tietyillä akuutissa myelooisessa leukemiassa esiintyvillä TET2-mutanteilla on heikentynyt kyky sitoa 2-oksoglutaraattia tai rautaa, mikä johtaa entsyymien aktiivisuuden laskuun. Kasvainkudoksissa happipitoisuudet ovat usein matalia nopean kasvun ja puutteellisen verisuonituksen vuoksi. TET-entsyymit ja histonidemetylaasit vaativat happea katalysoimissaan reaktioissa. Määritimme TET-entsyymien ja monien histonidemetylaasien riippuvuutta hapesta ja osoitimme, että H3K27-histonidemetylaasi KDM6A on erittäin riippuvainen hapesta, mikä osoittaa, ettei se pysty toimimaan kasvaimissa ja kudoksissa, joissa happipitoisuudet ovat matalia. Huomasimme, että vähähappisissa olosuhteissa solujen H3K27 metylaatio on lisääntynyt, mikä johti erilaistumisen estymiseen soluissa. Tämä tutkimus paljasti uusia mekanismeja useista syövistä löytyneiden muuntuneiden DNA:n ja histonien metylaatiotasojen taustalla. Häiriintynyt DNA:n ja histonien metylaatio on aiemmin yhdistetty syöpien etenemiseen, erityisesti solujen erilaistumisen häiriintymisen kannalta. Tässä tutkimuksessa yhdistimme 2-oksoglutaraatista riippuvaisten entsyymien inhibition häiriintyneeseen DNA:n ja histonien metylaatioon, joka voi johtaa muuntuneeseen solujen erilaistumiseen ja lopulta lisääntyneeseen syöpien aggressiivisuuteen ja invasiivisuuteen.

Oxygen homeostasis is essential to the life of aerobes, which is regulated in humans by Hypoxia Inducible Factor-1α (HIF-1α). Under hypoxic conditions, HIF-1α transactivates over a hundred genes related angiogenesis, erythropoiesis, etc. HIF-1α level and function is regulated by four HIF hydroxylase enzymes: three isoforms of prolyl hydroxylase domain (PHD1, PHD2 and PHD3) and factor inhibiting HIF-1α (FIH). PHD2 is the focus of this research. PHD2 is a non-heme Fe(II) 2-oxoglutarate dependent dioxygenase, which controls HIF-1α levels by hydroxylating two proline residues within the ODD domain of HIF-1α, then the hydroxylated prolines are recognized by pVHL, which targets HIF-1α for proteasomal degradation. Under hypoxic conditions PHD2 cannot hydroxylate HIF-1α and its level rises in cells. The aims of this research include understanding how PHD2 chooses its substrate, how the O2 activation occurs, and how certain transition metals inhibit PHD2. Our results revealed that electrostatics play a role in substrate selectivity of PHD2 by provoking a change in the opening and closing rate of β2β3 loop for NODD and CODD substrates. Mutational studies of second coordination sphere residues combined with kinetic studies indicated that decarboxylation of 2OG is the slow step in the chemical mechanism. The removal of a hydrogen-bond by the Thr387aAla mutation revealed a rate 15 times faster than WT-PHD2 by making O2 a better nucleophile. Our results indicate that this hydrogen bonding is essential for proper O2 activation. Previous reports show that certain metals increase HIF-1α levels by inhibiting PHD2. However there are conflicts about how this inhibition occurs, either through metal replacement from the active site or metals binding to a different site causing inhibition. Our competitive and non-competitive kinetic assays showed different inhibition profiles. Under competitive conditions Zn2+, Co2+, Mn2+, and Cu2+ can bind to the enzyme active site and lead to inhibition but under non-competitive conditions Zn2+, Co2+, and Mn2+ partially inhibit PHD2 suggesting that these metals cannot displace the Fe2+ from the active site. XAS experiments with Zn2+ and Fe3+ indicate that Zn2+ binds to the surface of PHD2 in a six-coordinate manner composed of two Cys201, 208, His205, Tyr197 and two water ligands.