Metalloproteins account for at least half of all known proteins. Metal ions often facilitate chemical that are energetically and/or kinetically challenging. Metal ion-dependent proteins are responsible for a myriad of essential biological functions, including respiration, biosynthesis of essential amino acids, nitrogen fixation, oxygen transport, photosynthesis and metabolisms (e.g. glycolysis and citric acid cycle). Not surprisingly, a growing number of disorders (e.g. various cancers, phenylketonuria, Wilson’s disease) are associated with mutations in metalloenzymes. A general introduction of the importance of metals in biology is presented in chapter 1. This thesis is aimed at obtaining a greater understanding of the structure and function of three metalloenzymes, ketol acid reductoisomerase (KARI), purple acid phosphatase (PAP) and metallo β lactamase (MβL). Chapter 2 examines the structure and dynamics of plant KARI. KARI is an enzyme in the branched-chain amino acid (BCAA) biosynthesis pathway. KARI is a binuclear Mg2+ enzyme that catalyses the conversion of 2-acetolactate (AL) into (2R)-2,3-dihydroxy-3-isovalerate or 2-aceto-2-hydroxybutyrate into (2R, 3R)-2,3-dihydroxy-3-methylvalerate in the presence of NADPH. To date, the only reported structures for a plant KARI are those of the spinach enzyme-Mn2+-(phospho) ADP ribose-(2R,3R)-2,3-dihydroxy-3-methylvalerate complex and the spinach KARI-Mg2+-NADPH-N-hydroxy-N-isopropyloxamate complex, where N-hydroxy-N-isopropyloxamate (IpOHA) is a predicted transition-state analog. These studies demonstrate that the enzyme is consisted of two domains, N- domain and C- domain, with the active site at the interface of these domains. In this chapter, the structures of the rice KARI-Mg2+ and rice KARI-Mg2+-NADPH complexes were determined to 1.55 and 2.8 Å resolutions, respectively. Comparisons of all the available plant KARI structures have revealed several major differences. Firstly, the N-domain is rotated up to 15o relative to the C-domain, expanding the active site by up to 4 Å. Secondly, an α-helix in the C-domain that includes residues V510-T519 and forms part of the active site moves by ~ 3.9 Å upon binding of NADPH. Thirdly, the 15 C-terminal amino acid residues in the rice KARI-Mg2+ complex are disordered. In the rice KARI-Mg2+-NADPH complex and spinach KARI structures, many of the 15 residues bind to NADPH and the N-domain and cover the active site. Fourthly, the location of the metal ions within the active site can vary by up to 2.7 Å. The new structures have thus, led to the proposal of an induced-fit mechanism. In this proposed induced-fit mechanism, (i) substrate enters the active site, (ii) active site is closed during catalysis, and (iii) the opening of active site facilitates product release. PAP is also a binuclear metalloenzyme and is capable of utilizing a heterovalent active site to hydrolyse a broad range of phosphomonoester substrates. Chapter 3 examines the catalytic mechanism of PAP based on several new crystal structures. The red kidney bean PAP structure in complex in sulphate was determined to 2.4 Å. This sulphate-bound structure provides insight into the pre-catalytic phase of its reaction cycle. This stucture demonstrates the significance of an extensive hydrogen-bonding network in the second coordination in initial substrate binding and orientation prior to hydrolysis. Most importantly, the two metal ions, Fe3+ and Zn2+, are five-coordinate in this structure, with only one nucleophilic μ-hydroxide present in the metal-bridging position. In combination with kinetic, crystallographic and spectroscopic data, all PAP structures form the proposal of a comprehensive eight-step model for the catalytic mechanism of purple acid phosphatases in general. To date, no reliable method for producing recombinant PAP at levels suitable for structural biology have been reported. Natural sources are the only way so far to obtaining PAP in a large quantity. Attempts to produce active and recombinant PAP from Mycobacterium marinum using bacterial are found in chapter 4. In brief, in combination with Nus fusion tag, Rosetta (DE3) strain and lower temperature (e.g. 25oC), expression of soluble and mycobacterial PAP becomes possible. However, this soluble protein is non-functional and thus, switching into other expression system (e.g.algal sytem) is the only approach to obtain soluble and functional protein. In algal expression system, human PAP was attempted. Preliminary results indicate that some PAP activity was observed when expressed in algal system. Chapter 5 focuses on the investigation of metallo β lactamase (MβL) from Klebsiella pneumoniae (Kp-MβL). This enzyme requires one or two Zn2+ ions for catalysis. Kinetic properties of Kp-MβL for the hydrolysis of various β-lactam substrates (e.g. benzyl-penicillin, cefoxitin, imipenem and meropenem) were investigated and the role of the metal ions in catalysis was also examined. Kinetic data demonstrate that Klebsiella pneumoniae MβL can degrade a broad spectrum of β-lactam antibiotics, with a high preference for cephems and carbapenems. Kinetic data from pH dependence studies has revealed that catalysis of benzyl-penicillin and meropenem is preferred at acidic pH. The kcat vs pH profile demonstrates that catalysis is enhanced by protonation, thus it is likely that the relevant group is responsible for the donation of a proton to the product or leaving group. In this case, a doubly Lewis activated, bridging hydroxide molecule has been speculated. A single protonation event (pKa ~7) is also observed in kcat/Km vs pH profile. Since benzyl-penicillin does not have an acidic moiety in this pH range, this event is likely to be associated with the free enzyme. His 79 and 139 have been speculated to enhance substrate binding. In contrast, catalysis of both cefoxitin and imipenem is favoured at alkaline pH, leading to the proposal that a terminally bound water is likely to form a nucleophile. A bell-shaped pH profile for kcat/Km is observed for cefoxitin and imipenem substrates. pKa of ~ 9-9.5 is likely to be associated with Lys161, which enhances substrate binding. In Chapter 6, a novel MβL from Serratia proteamaculans (Spr-MβL) is investigated. This chapter includes expression, purification and preliminary characterization of this MβL using steady-state kinetics. Expression of this enzyme in Rosetta (DE3) plysS E. coli strain yields only a small amount of soluble enzyme (1 mg/ 6 L culture). To improve the amount of soluble protein, Spr-MβL was subjected to several rounds of in vitro evolution. About two-fold gain in solubility was achieved by this method along with a five-fold increase in β-lactamase activity. Further rounds of directed evolution are now planned. The kinetic behaviour for Spr-MβL-catalysed the hydrolysis of three β-lactam substrates, penicillin, cefoxitin and imipenem were also studied. Kinetic data suggest that a water molecule bridging the two Zn2+ ions is the likely nucleophile in the reaction with penicillin while the reaction-initiating nucleophile is likely to be a terminally bound hydroxide in the reaction with cephalothin and imipenem (Chapter 6). In summary, this project has led to a better understanding of the structures of KARI and PAP prior to catalysis. This project has also aided in the understanding of catalytic mechanism of MβLs and the role the metal ions play. The knowledge gained will facilitate the development of new chemotherapeutics and herbicides.

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Sous l'effet du stress oxydatif, la production des espèces réactives de l'oxygène (ERO) est accrue. Les ERO peuvent réagir avec différentes molécules biologiques dont les acides gras polyinsaturés libres ou provenant des lipides membranaires engendrant la formation d'hydroperoxydes d'acides gras. Dans le chloroplaste, ces molécules sont sujettes à de nombreuses modifications enzymatiques ou chimiques aboutissant à la formation d'oxylipines. Les oxylipines participent à la signalisation cellulaire (précurseurs de la voie du jasmonate par exemple), à la défense de la plante (activité antimicrobienne par exemple). Certaines de ces molécules très réactives et toxiques doivent être métabolisées en des produits moins réactifs, par des réactions d'oxydoréduction par exemple.La ceQORH (chloroplast envelope Quinone OxydoReductase Homolog) et IEP32 (Inner Envelope Protein 32) d'Arabidopsis thaliana sont des oxydoréductases chloroplastiques impliquées dans la détoxification de la plante. Elles sont transportées au travers de l'enveloppe du chloroplaste sans clivage de leur peptide de transit par une voie d'import alternative à la voie TOC/TIC (Translocon at the Outer envelope membrane of Chloroplasts et Translocon at the Inner envelope membrane of Chloroplasts). Ces particularités ont fait de la ceQORH et d'IEP32 des enzymes intéressantes à étudier.IEP32 a été purifiée et cristallisée. Les structures de la ceQORH sous forme apo, liée au NADPH et liée au NADP+ et à des inhibiteurs ont été déterminées en utilisant la cristallographie aux rayons X. Leur analyse a permis de mettre en évidence que la ceQORH existe sous différents états oligomériques dans les conditions expérimentales utilisées. Ces observations ont été confirmées par des expériences d'ultracentrifugation analytique. La ceQORH est une enzyme monomérique qui lie le NADPH par l'intermédiaire de son domaine de Rossmann . Le site catalytique, large et hydrophobe, permet à la ceQORH d'accommoder et de réduire un grand nombre de substrat dont le motif commun est la présence d'une fonction alcène en α, β d'un groupement carbonyle. Les constantes d'efficacité et d'affinité étant meilleures pour les γ-kétols que pour les quinones, nous proposons que la ceQORH soit renommée « γ-kétol réductase ».Mots clefs : chloroplaste - Medium chain dehydrogenase/reductase - γ-kétol réductase - oxylipines - oligomérisation - inhibition - cristallographie aux rayons X - ultracentrifugation analytique / Under the influence of the oxidative stress, the production of the Reactive Oxygen Species (ROS) is increased. These compounds can react with various biological molecules like free polyunsaturated fatty acids or derived from lipids producting hydroperoxides of fatty acids. In chloroplast, these molecules are subject to numerous enzymatic or chemical modifications resulting in oxylipins. Oxylipins participate in cell signaling (precursors of the way of the jasmonate for example), or in the defense of the plant (antimicrobial activity). However some of them are very reactive and toxic so they are metabolized in less reactive molecules by oxidoreduction reactions.The ceQORH (chloroplast envelope Quinone OxydoReductase Homolog) and IEP32 (Inner Envelope Protein 32) of Arabidopsis thaliana are chloroplast oxidoreductases involved in the plant detoxification. They are transported through the envelope of the chloroplast without cleavage of their transit peptide by an alternative import pathway of TOC/TIC (Translocon at the Outer envelope membrane of Chloroplasts and Translocon at the Inner envelope membrane of Chloroplasts). In order to better understand their roles, we studied their enzymatic properties and structures.IEP32 was purified and crystallized. The structures apo-ceQORH and bound to the NADPH/ NADP + with inhibitors were determined using X-ray crystallography. The analysis allowed us to show that ceQORH exists under different oligomerization states what was confirmed by results of analytical ultracentrifugation. The NADPH binding to the Rossmann fold induces the monomerization. The catalytic site is large and hydrophobic allowing to ceQORH to reduce many α, β-unsaturated carbonyls of various chain lengths. CeQORH was shown to reduce with high efficiency the reactive double bond of γ-ketols that's why we propose to rename ceQORH by “γ- ketol reductase”.Keywords: chloroplast – Medium chain dehydrogenase/reductase – γ-ketol reductase – oxylipins – oligomerization state – inhibition – X-ray crystallography – analytical ultracentrifugation