Quantum chemistry has nowadays become a powerful and efficient tool that can be successfully used for studies of biosystems. It is therefore possibleto model the enzyme active-site and the reactions undergoing into it, as well as obtaining quite accurate energetic profiles. Important conclusions can be drawn from such profiles about the  plausibility of different putative mechanisms. Density Functional Theory is used in the present thesis for investigation of the catalytic mechanism of dioxygenase metallo-enzymes and synthetic model complexes. Three enzymes were studied – Homoprotocatechuate 2,3-dioxygenase isolated from Brevibacterium fuscum (Bf 2,3-HPCD), Manganese-Dependent Homoprotocatechuate 2,3-Dioxygenase (MndD) and Homogentisate Dioxygenase (HGD). Models consisting of 55 to 208 atoms have been built from X-ray crystal structures and used in the calculations. The computed energies were put in energy curves and were used for estimation of the feasibility of the suggested reaction mechanisms. A non-heme [(L4Me4)Fe(III)]+3 complex that mimics the reactivity of intradiol dioxygenases, and a heme [T(o-Cl)PPFe] complex catalyzing the stepwise oxidation of cyclohexane to adipic acid, were also studied. For the enzymes and the non-heme biomimetic complex the reaction was found to follow a mechanism that was previously suggested for extradiol and intradiol dioxygenases – ordered substrates binding and formation of peroxo species, which further undergoes homolytic O-O bond cleavage. Different reaction steps appear to be rate limiting in the particular cases: proton transfer from the substrate to the peroxide in Bf 2,3-HPCD, the formation of the peroxo bridge in HGD and the biomimetic complex, and notably, spin transition in MndD. The catalytic oxidation of cyclohexane to adipic acid in the presence of molecular oxygen as oxidant was studied, a reaction of great importance for the chemical industry. Reaction mechanism is suggested, involving several consecutive oxidative steps. The highest calculated enthalpy of activation is 17.8 kcal/mol for the second oxidative step. / At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 4: In progress, Paper 5: In progress

Neste trabalho, usamos a técnica de Ressonância Paramagnética Eletrônica em seu modo convencional para o estudo da enzima clorocatecol 1,2-dioxigenase e em seu modo pulsado para o estudo da estrutura dinâmica de biomembranas. Clorocatecol 1,2-dioxigenase é uma enzima que catalisa a clivagem de estruturas aromáticas, como a do clorocatecol, via a a ativação e incorporação de uma molécula de oxigênio e que possui em seu sítio ativo um íon Fe(III). Nossos resultados de CD e atividade enzimática mostram o intervalo de temperaturas entre 20-25 ºC como aquele no qual a enzima apresenta atividade máxima. A maior contribuição para sua estrutura secundária vem de folhas β anti-paralela. A quantificação do número de spin feita por EPR indicou a presença de um íon Fe(III) por molécula de CCD, estando esse íon em estado de spin alto e simetria rômbica, como se depreende da linha estreita em g=4,3. Os parâmetros de desdobramentos de campo zero foram determinados pela medida em função da temperatura da linha em g=4,3, fornecendo λ=E/D=1/3 e D=(1,3±0,2) cm -1. O gráfico de Scatchard construído a partir dos espectros de EPR quando da titulação da enzima com o substrato catecol indicou a presença de um único sítio ligante de catecol, em acordo com a quantificação anterior do metal, e cuja constante de afinidade enzima-substrato é k=(2,7±0,1)x10-6 M. No que tange a EPR pulsado, foram utilizadas as chamadas técnicas modernas de EPR, com ênfase em 2D-ELDOR, para a investigação de diversos aspectos em membranas de interesse biológico. Primeiramente, investigamos a diferenciação entre a fase de líquido ordenado (Lo) e a de líquido cristalino (Lc) em membranas modelo de lipídio puro e lipídio/colesterol (1/1) contendo diferentes marcadores de spin (16-PC, CSL e DPPTC). Aí mostramos que é possível distinguir-se diferentes fases lipídicas apenas por simples inspeção visual dos espectros de 2D-ELDOR de um determinado marcador de spin incorporado à membrana. Além disso, realizamos simulações através do pacote de programas NLSPMC e determinamos as diferenças quantitativas entre as fases lipídicas: o estado de líquido ordenado apresenta maior fluidez e ordenamento na região das cadeias acílicas, ao passo que, na região da cabeça polar, mostra menor ordenamento. Em seguida, estudamos o efeito da presença de colesterol em alta concentração sobre o comportamento da membrana como função da temperatura. Nesse caso, o colesterol atua de maneira a manter a membrana em uma fase altamente ordenada e fluída dentro do intervalo de temperaturas medido, abolindo a transição gel-Lc do lipídio DPPC. Uma tentativa de aplicação de nossa metodologia ao estudo de membranas biológicas foi feita ao estudarmos membranas bleb que são ricas em colesterol. Estas mostram comportamento semelhante àquele observado para as membranas modelo com alta concentração de colesterol, um indicativo da existência de domínios Lo naquelas membranas biológicas. Por fim, estudamos o efeito da presença do peptídeo Gramicidina A\' (GA) sobre a estrutura da membrana de DPPC. Os resultados de 2DELDOR mostram claramente a presença de duas populações de lipídios (\"boundary\" e \"bulk\"). As simulações desses espectros foram as primeiras feitas com espectros contendo duas componentes e com os dados nas formas Sc- e Secsy. Essas mostram que os lipídios \"bulk\" são pouco afetados pela presença de moléculas de GA, ao passo que os lipídios \"boundary\" têm suas cadeias acílicas dobradas na porção terminal em torno das moléculas de GA, mecanismo que suporta a formação de domínios em fase HII na membrana. / In this work, EPR-CW and 2D-FT-EPR are used to study the enzyme chlorocatechol 1,2-dioxygenase and the dynamic structure of biological relevant membranes, respectively. Chlorocatechol 1,2-dioxygenase (CCD) is a non-heme Fe(lIl) enzyme that catalyses the ring cleavage of aromatic compounds like chlorocatechol. The structure stability as a function of the temperature was determined via circular dichroism and catalytic activity assays. The enzyme has its maximum activity at 20-25 ºC. The main contribution to its secondary structure comes from antiparallel Β sheets. The iron content was determined by EPR measurements, indicating the presence of one Fe(llI) per molecule. The Fe(III) ion shows a very narrow line at g=4.3 indicating a high spin state in a rhombic symmetry with λ=E/D=1/3 and D=(1,3±0,2) cm -1. The Scatchard plot based on EPR spectra suggests the existence of one site for substrate binding with a binding constant k=(2,7±0,1)x10-6 M. As for 2D-FT-EPR, the so-called modem EPR techniques, like 2D-ELDOR, were used to investigate several aspects of biologically relevant membranes. Firstly, we determined the differences between the liquid-ordered (Lo) and the liquid-crystalline (Lc) phases in model membranes of pure lipid and of lipid/cholesterol (1/1) mixtures containing different spin labels (16-PC, CSL, and DPPTC). In this case, we show how 2D-ELDOR makes possible the differentiation between Lo and Lc phases just by a pattern recognition scheme. We also performed simulations of those spectra and the results are: the Lo phase shows higher fluidity and ordering in the acyl chain region, whereas it shows lower ordering in the headgroup polar region. After that the membrane behaviour as s function of temperature was studied. We showed that cholesterol maintains the membrane in a highly ordered and fluid structure over the entire range of temperatures, abolishing the gel-Lc phase transition of the lipid DPPC. The biological membrane bleb was the first attempt to apply our methodology to real membranes. The 2D-ELDOR results for bleb membranes indicate the existence of Lo domains in the structure of those membranes. Finally, we studied the effects of the peptide Gramicidin A\' (GA) on the lipid organization of DPPC membranes. The 2D-ELDOR results show very clear two-component spectra (assigned to bulk and ,boundary lipids), which were simulated by the NLSPMC programs. This is the first time that 2D-FT-EPR multi-component spectra are simulated using both the Sc- and Secsy formal. The simulations indicate that the GA molecules do not significantly affect the bulk lipid, whereas the boundary lipids present the end portion of their acyl chain bent towards the GA molecule. This mechanism is probably responsible for the local formation of the HII phase in the membranes.

Yes / Execution of lineage-specific differentiation programs requires tight coordination between many regulators including Ten-eleven translocation (TET) family enzymes, catalyzing 5-methylcytosine oxidation in DNA. Here, by using Keratin 14–Cre–driven ablation of Tet genes in skin epithelial cells, we demonstrate that ablation of Tet2/Tet3 results in marked alterations of hair shape and length followed by hair loss. We show that, through DNA demethylation, Tet2/Tet3 control chromatin accessibility and Dlx3 binding and promoter activity of the Krt25 and Krt28 genes regulating hair shape, as well as regulate interactions between the Krt28 gene promoter and distal enhancer. Moreover, Tet2/Tet3 also control three-dimensional chromatin topology in Keratin type I/II gene loci via DNA methylation–independent mechanisms. These data demonstrate the essential roles for Tet2/3 in establishment of lineage-specific gene expression program and control of Dlx3/Krt25/Krt28 axis in hair follicle epithelial cells and implicate modulation of DNA methylation as a novel approach for hair growth control. / This work was supported by the National Institutes of Health grant 5R01 AR075776 (V.A.B. and A.A.S.) and grant 5R01 AR071727 (V.A.B. and A.A.S.) and the National Science Foundation of China (G.-L.X.). / Research Development Fund Publication Prize Award winner, Dec 2022.

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

Organoarsenicals such as methylarsenical methylarsenate (MAs(V)) and aromatic arsenicals including roxarsone (4-hydroxy-3-nitrophenylarsenate or Rox(V)) have been extensively used as an herbicide and growth enhancers in animal husbandry, respectively. They undergo environmental degradation to more toxic inorganic arsenite (As(III)) that contaminates crops and drinking water. We previously identified a bacterial gene (arsI) responsible for aerobic MAs(III) demethylation. The gene product, ArsI, is a Fe(II)-dependent extradiol dioxygenase that cleaves the carbon-arsenic (C-As) bond in MAs(III) and trivalent aromatic arsenicals. The objective of this study was to elucidate the ArsI mechanism. Using isothermal titration calorimetry, we determined the dissociation constants (Kd) and ligand-to-protein stoichiometries (N) of ArsI for Fe(II), MAs(III) and aromatic phenyl arsenite. Using a combination of methods including chemical modification, site-directed mutagenesis, and fluorescent spectroscopy, we demonstrated that amino acid residues predicted to participate in Fe(II)-binding (His5-His62-Glu115) and substrate binding (Cys96-Cys97) are all involved in catalysis. Finally, the products of Rox(III) degradation were identified as As(III) and 4-hydroxy-2-nitrophenol, demonstrating that ArsI is a dioxygenase that incorporates one oxygen atom from dioxygen into the carbon and the other to the arsenic to catalyze the cleavage of the C-As bond. These results augment our understanding of the mechanism of this novel C-As lyase.

Afin d’optimiser la production d’un composé d’intérêt tout en évitant les conséquences néfastes sur la viabilité cellulaire, un défi majeur de l’ingénierie métabolique est d’obtenir un équilibre entre les flux métaboliques endogènes de la cellule et le flux consommé par une nouvelle voie de biosynthèse. Dans ce contexte d’optimisation, les stratégies combinatoires génèrent une diversité de voies métaboliques et de modes de régulation rassemblant de précieuses informations quant aux choix d’orientations stratégiques à faire. Notre étude s’inscrit dans un projet visant à produire les molécules responsables de l’arôme, de la couleur et du parfum du safran (Crocus sativus) chez Saccharomyces cerevisiae. Une approche combinatoire a été adoptée pour moduler les niveaux d’expression de trois gènes menant à la synthèse de leur précurseur commun : la zéaxanthine. Cette stratégie nous a permis de décrire des biais inattendus dans la régulation des niveaux d’expression des gènes exprimés sur plasmide. Nous avons détecté une forte interférence transcriptionnelle entre les gènes dans notre système, ainsi qu’une influence de la nature des séquences codantes. Ces éléments, identifiés comme critiques, imposent sur les niveaux d’expression des trois gènes une régulation plus importante que la force des promoteurs qui les contrôlent. Afin de poursuivre le projet vers son objectif final, la réaction de clivage de la zéaxanthine menant à la synthèse des composés d’intérêt du safran a fait l’objet d’une analyse fonctionnelle détaillée. Une absence d’activité de l’enzyme décrite dans la littérature comme responsable de cette réaction nous a conduits à proposer des perspectives d’ingénierie pour atteindre l’objectif final du projet / To optimize the production of a value added compound while avoiding toxic consequences on the cell viability, a challenge in the metabolic engineering field is to balance the endogenous metabolic fluxes and the newly consumed fluxes. In this optimization context, combinatorial strategies can generate several variants of synthetic metabolic pathways. This strategy gives precious strategic information on the right combinations of function and regulation choices to be made in the ultimate pathway reconstruction. Our study aimed at the production of the molecules responsible for aroma, dye, and fragrance of saffron (Crocus sativus) in Saccharomyces cerevisiae. A combinatorial approach was chosen to modulate expression levels of three genes involved in their common precursor biosynthesis: zeaxanthin. This strategy allowed us to describe some unexpected bias in the regulation of the plasmid-encoded genes expression levels. We detected strong transcriptional interference between the different genes in our system, and the ORF nature also seemed to influence the expression levels. These critical factors imposed a stronger regulation of the three genes expression levels than the promoter strength initially chosen to control them. The project was continued toward its final objective by making a detailed functional analysis of the zeaxanthin cleavage reaction leading to the molecules of interest synthesis. This reaction was described to be catalyzed by a specific enzyme, but no activity was observed in our experiments. This result led us to propose some tools to reach the final goal of the project

Hypoxia-inducible factor (HIF) is the master regulator of genes involved in adaptation to hypoxia. The stability and transcriptional activity of HIF are regulated by post-translational hydroxylations: prolyl hydroxylation by the prolyl hydroxylase domain-containing enzymes PHD1 – 3 earmarks HIF for proteasomal degradation, whilst asparaginyl hydroxylation by factor inhibiting HIF (FIH) blocks the interaction of HIF with the transcriptional coactivators p300/CBP. The PHDs and FIH hydroxylate HIF directly from molecular oxygen and are therefore oxygen sensors. Recent literature shows that FIH also hydroxylates a number of proteins containing an ankyrin-repeat domain (ARD). Together with reports suggesting that the PHDs are involved in HIF-independent pathways, this suggests that the HIF hydroxylases may have a wide range of non-HIF targets. This thesis describes my investigations into novel substrates of the HIF hydroxylases. This work has characterized the FIH-dependent hydroxylation of the ARD-containing protein Notch1, and defined a consensus sequence for hydroxylation that corresponds to the ankyrin-repeat consensus. Using this consensus potential sites of hydroxylation in a novel ARD FIH substrate, myosin phosphatase targeting subunit 1 (MYPT1), were identified then subsequently confirmed and characterized. Notch1 competes with HIF for FIH hydroxylation. My experiments show that this occurs because Notch1 is a more efficient substrate than HIF, whilst studies on MYPT1 and other proteins indicate that competitive inhibition of FIH may be a general property of ARDs. There are more than 300 ARD proteins in the human genome, and this thesis demonstrates that FIH may hydroxylate a significant percentage of these. In addition to the analysis of ARD hydroxylation a proteomic investigation into novel PHD3 substrates has identified two candidate proteins, suggesting that the PHDs may also have multiple targets. These results have important implications for oxygen sensing, and indicate that post-translational hydroxylation is likely to be a widespread modification in cell biology.

Les hydrocarbures aromatiques polycycliques (HAP) sont des composés ubiquitaires issus de la combustion incomplète de matières organiques. Ils sont à l'origine de pollutions de l'environnement, surtout liées à l'exploitation des produits pétroliers, car ce sont des composés toxiques pour les êtres vivants et pour l'homme en particulier. De nombreuses bactéries capables de dégrader les HAP ont été isolées et étudiées, mais celles qui les dégradent in situ sont mal connues, car moins de 5% des bactéries du sol sont cultivables en laboratoire. Le premier objectif de cette étude était d'identifier les bactéries qui dégradent les HAP dans le sol par des méthodes moléculaires indépendantes de la culture. A cette fin, une stratégie de marquage isotopique in situ a été mise en œuvre qui repose sur l'utilisation du phénanthrène, un HAP à trois cycles, dans lequel l'isotope naturel du carbone a été remplacé par le 13C. Cette molécule a été introduite comme traceur dans des microcosmes contenant du sol provenant d'un bassin de rétention des eaux de ruissellement d'autoroute. Les bactéries ayant incorporé le 13C ont ensuite été identifiées par séquençage des gènes d'ARNr 16S amplifiés à partir de l'ADN marqué extrait du sol. Les résultats montrent que des Betaprotéobactéries peu étudiées à ce jour, appartenant aux genres Acidovorax, Rhodoferax, Hydrogenophaga et Thiobacillus, ainsi que des Rhodocyclaceae, étaient les principaux acteurs de la dégradation du phénanthrène. La prépondérance des Betaprotéobactéries a été établie par des mesures de PCR quantitative. Une analyse dynamique de la diversité bactérienne a montré que celle-ci changeait en fonction de la biodisponibilité du phénanthrène. En outre, la diversité d'arène-dioxygénases impliquées dans la dégradation des HAP a été explorée sur le plan phylogénétique et fonctionnel. Nous avons ainsi détecté des séquences nouvelles, pour la plupart apparentées à des dioxygénases de Sphingomonadales et de Burkholderiales. Grâce à la construction et l'expression d'enzymes hybrides, il a été possible, pour la première fois, d'associer une activité catalytique d'oxydation des HAP à des séquences partielles de gènes, amplifiées à partir de l'ADN du sol. Les résultats obtenus et les outils mis au point dans cette étude pourront servir à développer des méthodes de diagnostic et de suivi de biodégradation de polluants, par exemple dans le cadre d'opérations de bioremédiation de sites pollués par les HAP. / Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous compounds produced by incomplete combustion of organic matter. They are a source of environmental pollution, especially associated to oil product exploitation, and represent a threat for living organisms including human beings because of their toxicity. Many bacteria capable of degrading PAHs have been isolated and studied. However, since less than 5% of soil bacteria can be cultivated in the laboratory, bacterial species able to degrade PAHs in situ have been poorly studied. The first goal of this study was to identify bacteria that degrade PAHs in soil using culture-independent molecular methods. To this end, a strategy known a stable isotope probing has been implemented based on the use of phenanthrene, a three rings PAH, in which the natural isotope of carbon was replaced by 13C. This molecule has been introduced as a tracer in microcosms containing soil from a constructed wetlands collecting contaminated water from highway runoff. Bacteria having incorporated the 13C were then identified by 16S rRNA gene sequence analysis after PCR amplification from labeled genomic DNA extracted from soil. The results show that so far little studied Betaproteobacteria, belonging to the genera Acidovorax, Rhodoferax, Hydrogenophaga and Thiobacillus, as well as Rhodocyclaceae, were the key players in phenanthrene degradation. Predominance of Betaprotéobactéries was established thanks to quantitative PCR measurements. A dynamic analysis of bacterial diversity also showed that the community structure of degraders depended on phenanthrene bioavailability. In addition, the phylogenetic diversity of ring-hydroxylating dioxygenases, enzymes involved in the first step of PAH degradation, has been explored. We detected new sequences, mostly related to dioxygenases from Sphingomonadales and Burkholderiales. For the first time, we were able to associate a catalytic activity for oxidation of PAHs to partial gene sequences amplified from soil DNA, by constructing hybrid enzymes and assaying their activity The results obtained and the tools implemented in this study may be used to develop methods for the diagnostic and monitoring of pollutant biodegradation in processes such as bioremediation of PAHs contaminated sites.