Rozprawy doktorskie na temat „Zinc metalloenzyme”

Escherichia coli 5'-nucleotidase is a periplasmically localised enzyme capable of hydrolysing a broad range of substrates, including all 5'-ribo- and 5'-deoxyribonucleotides, uridine diphosphate sugars, and a number of synthetic substrates such as bis (r-nitrophenyl) phosphate. The enzyme has been shown to contain at least one zinc ion following purification, and to have two metal binding sites in the catalytic cleft. 5'-Nucleotidase activity is significantly stimulated by the addition of particular divalent metal ions, most notably cobalt which results in a 30-50 fold increase in activity. Significant sequence homology between the E. coli 5'-nucleotidase and members of the Ser/Thr protein phosphatase family in the catalytic site has lead to 5'-nucleotidase being included in this protein family. This thesis describes the development of a rapid purification methodology for milligram quantities of 5'-nucleotidase, and the investigation of a number of physical and biochemical properties of the enzyme with the aim of comparing these properties to those of certain catalytic site mutants. The molecular weight of the mature protein was estimated as 58219 daltons, with a specific activity for 5'-AMP, in the presence of 4 mM Co2+ and 13 mM Ca2+ at pH 6.0, of 730 mmol/min/mg. The presence of up to two zinc ions associated with the purified enzyme was observed using ICP-ES analysis, suggesting both metal ion binding sites are occupied by zinc in vivo, and some degree of displacement of zinc by cobalt could be observed. Mass spectrometry data, gathered at 60 and 70 mS orifice potential, suggested the presence of a small proportion of material with a mass 118 to 130 daltons greater than the main 5'-nucleotidase mass estimation. This study suggests that this mass difference, only evident at the lower orificepotential, is due to the presence of two zinc ions closely associated with 5'-nucleotidase. To account for the observed high level of activation of 5'-nucleotidase activity by particular divalent metal ions, this thesis describes a proposed model in which these divalent ions may displace the zinc ion at one of the metal ion binding sites. This displacement only occurs at one of the two metal ion binding sites, with the other metal binding site retaining the zinc ion already present. Studies with purified enzyme, each with a single amino acid substitution, lend support to this hypothesis and suggest the identity of the metal ion binding site at which displacement occurs. Seven key catalytic site residues (Asp-41, His-43, Asp-84, His-117, Glu-118, His-217 and His-252) were selected on the basis of sequence conservation within the Ser/Thr protein phosphatases and 5'-nucleotidases. X-ray crystallographic data published by others during this study implicated five of the selected residues (Asp-41, His-43, Asp-84, His-217 and His-252) directly in metal ion binding, including two residues from each metal ion binding site and one directly involved in both sites (Asp-84). The remaining two residues (His-117 and Glu-118) are highly conserved but were not thought to play direct roles in metal ion binding. The seven selected residues were modified by site-directed mutagenesis, and the effect of the amino acid substitutions upon the kinetic properties of 5'-nucleotidase activity was determined. Residues hypothesised to be involved in metal ion displacement, and subsequent activation of 5'-nucleotidase activity, were identified by reductions in metal ion affinity and increased levels of activation by cobalt compared to the wild type 5'-nucleotidase. This study suggests that the metal binding site, M2, that includes residues Asp-84, His-217 and His-252, is involved in metal ion displacement, while the other metal binding site, M1, is not. This, in turn, suggests the metal binding sites are functionally non-equivalent and kinetically distinct. No residues were identified in this study as playing significant roles in substrate binding, as there was no significant reduction observed in affinity for 5'-AMP observed in any of the catalytic site mutants.

Escherichia coli 5'-nucleotidase is a periplasmically localised enzyme capable of hydrolysing a broad range of substrates, including all 5'-ribo- and 5'-deoxyribonucleotides, uridine diphosphate sugars, and a number of synthetic substrates such as bis (r-nitrophenyl) phosphate. The enzyme has been shown to contain at least one zinc ion following purification, and to have two metal binding sites in the catalytic cleft. 5'-Nucleotidase activity is significantly stimulated by the addition of particular divalent metal ions, most notably cobalt which results in a 30-50 fold increase in activity. Significant sequence homology between the E. coli 5'-nucleotidase and members of the Ser/Thr protein phosphatase family in the catalytic site has lead to 5'-nucleotidase being included in this protein family. This thesis describes the development of a rapid purification methodology for milligram quantities of 5'-nucleotidase, and the investigation of a number of physical and biochemical properties of the enzyme with the aim of comparing these properties to those of certain catalytic site mutants. The molecular weight of the mature protein was estimated as 58219 daltons, with a specific activity for 5'-AMP, in the presence of 4 mM Co2+ and 13 mM Ca2+ at pH 6.0, of 730 mmol/min/mg. The presence of up to two zinc ions associated with the purified enzyme was observed using ICP-ES analysis, suggesting both metal ion binding sites are occupied by zinc in vivo, and some degree of displacement of zinc by cobalt could be observed. Mass spectrometry data, gathered at 60 and 70 mS orifice potential, suggested the presence of a small proportion of material with a mass 118 to 130 daltons greater than the main 5'-nucleotidase mass estimation. This study suggests that this mass difference, only evident at the lower orificepotential, is due to the presence of two zinc ions closely associated with 5'-nucleotidase. To account for the observed high level of activation of 5'-nucleotidase activity by particular divalent metal ions, this thesis describes a proposed model in which these divalent ions may displace the zinc ion at one of the metal ion binding sites. This displacement only occurs at one of the two metal ion binding sites, with the other metal binding site retaining the zinc ion already present. Studies with purified enzyme, each with a single amino acid substitution, lend support to this hypothesis and suggest the identity of the metal ion binding site at which displacement occurs. Seven key catalytic site residues (Asp-41, His-43, Asp-84, His-117, Glu-118, His-217 and His-252) were selected on the basis of sequence conservation within the Ser/Thr protein phosphatases and 5'-nucleotidases. X-ray crystallographic data published by others during this study implicated five of the selected residues (Asp-41, His-43, Asp-84, His-217 and His-252) directly in metal ion binding, including two residues from each metal ion binding site and one directly involved in both sites (Asp-84). The remaining two residues (His-117 and Glu-118) are highly conserved but were not thought to play direct roles in metal ion binding. The seven selected residues were modified by site-directed mutagenesis, and the effect of the amino acid substitutions upon the kinetic properties of 5'-nucleotidase activity was determined. Residues hypothesised to be involved in metal ion displacement, and subsequent activation of 5'-nucleotidase activity, were identified by reductions in metal ion affinity and increased levels of activation by cobalt compared to the wild type 5'-nucleotidase. This study suggests that the metal binding site, M2, that includes residues Asp-84, His-217 and His-252, is involved in metal ion displacement, while the other metal binding site, M1, is not. This, in turn, suggests the metal binding sites are functionally non-equivalent and kinetically distinct. No residues were identified in this study as playing significant roles in substrate binding, as there was no significant reduction observed in affinity for 5'-AMP observed in any of the catalytic site mutants.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Biomolecular and Biomedical Sciences
Science, Environment, Engineering and Technology
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Les anticorps catalytiques associent les propriétés de reconnaissance des anticorps aux propriétés de catalyse des enzymes. Différentes approches ont été envisagées pour leur faire mimer les enzymes : analogues d'états de transition, copie du site actif par le réseau idiotypique, ingénierie des protéines. De nombreuses enzymes s'associent avec des métaux pour assurer leurs fonctions. Le zinc est un élément intéressant dans ce cas car retrouvé dans plus de 300 enzymes. Ces sites de fixation étant bien caractérisés, ils ont pu semir de modèle en biotechnologie dans l'ingénierie des protéines. L'approche que nous proposons est basée sur la biosynthèse de nove du site de fixation du zinc catalique. Pour cela, nous nous appuyons sur les règles de reconnaissance de l'IDA-Zn (Il) par les protéines (IMAC). L'immunisation d'un animal contre l'IDA-Zn(ll) devrait produire des anticorps présentant au niveau de leur paratope une structure de ligands capable de fixer un ion métallique dans une conformation catalytique. Pour développer ce type d'anticorps, une nouvelle méthode ELISA a d'abord été développée. L'utilisation d'un présentateur dIhaptène non protéique (le PEG bifonctionalisé) nous permet d'éviter toute réaction croisée avec la protéine de trans ort utilisée pour l'immunisation. Les anticorps anti-IDA-Zn(ll) ont ensuite été réalisés selon deux tech¬niques. D'abord la méthode des hybridomes nous a permis d'isoler à partir de 1152 clones 14 clones présentant de bonnes affinités. Ensuite, le répertoire immunologique de la souris immunisée contre l'IDA-Zn(ll) a été exprimé en banque de phages (banque de scFv). L'avantage d'une telle banque réside dans l'expression de tout le répertoire immunologique de la souris immunisée, ce qui offre la possibilité de "screener" des spécificités plus vastes vis-à-vis d'autres métaux, chélates métalliques ou d'autres antigènes. Enfin, la production de ces anticorps anti-chélate métallique à grande échelle nous a fait apparaître la nécessité de développer une méthode de purification douce, respectueuse de la structure tridimensionnelle des anticorps. Une méthode, faisant appel au ligand pseudobiospécifique l'histidine, a été développée au laboratoire. Pour valider son utilisation aux anticorps catalytiques, nous avons décidé de l'appliquer à la purification des anticorps catalytiques naturels présents dans le sérum des malades afteints de maladies auto-immunes. Les résultats sont comparés avec les méthodes classiques protéine A et protéine G, en termes de pureté des fractions et d'activité catalytique.

Histone deacetylases (HDACs) are a class of enzymes that play a crucial role in DNA expression by removing an acetyl group from the ɛ-N-acetyl lysine residue on histone proteins. Out of 18 isoforms of HDAC enzymes which are classified into 4 classes, only 11 of them are metalloenzymes that require zinc for its catalytic activity. HDACs are considered promising target for drug development in cancer and other parasitic diseases due to their role in gene expression. Histone deacetylase inhibitors (HDACi) can cause cell cycle arrest, and induce differentiation or apotosis. While HDACi shows promising antitumor effects, their mechanism of action and selectivity against cancer cells have not been adequately defined yet. In addition, low oral bioavailability, short half-life time, bone marrow toxicity, and cardiotoxicity limit their use in clinic. Therefore, there is considerable interest in developing compounds with selectivity and specificity towards individual family members of HDACs. The prototypical pharmacophore for HDAC inhibitors consist of a metal-binding moiety that coordinates to the catalytic metal ion within the HDAC active site, a capping group that interacts with the residues at the entrance of the active site and a linker that appropriately positions the metal-binding moiety and capping group for interactions in the active site. It has been shown that modification of cap, cap linking moiety, linker or zinc binding group (ZBG) shows promises of superior potency and isoform selectivity. My thesis research involves manipulating different aspects of the pharmacophoric model to yield not only more potent, selective, and effective drugs but also to help understand the biology of HDAC isoforms. In addition, I was successful in extending studies on HDAC isoforms to other zinc metalloenzymes such as leishmanolysin (gp63) and spliceosome associated zinc-metalloenzymes to understand biology of these zinc metalloenzymes by developing potent and selective small molecule inhibitors. This will aid in improvement of existing therapeutics for treatment of cancer, leishmania, malaria and other genetic disorders.

La synthèse guidée par la cible de ligands protéiques est une stratégie innovante pour découvrir des composés bioactifs. En particulier, la Kinetic Target-Guided Synthesis (KTGS) and the Dynamic Combinatorial Chemistry (DCC) ont permis, ces dernières années, de découvrir des ligands originaux pour des cibles thérapeutiques mal explorées, ce qui a permis de lancer des projets de découverte de médicaments. Ce projet de thèse vise à utiliser la KTGS pour découvrir, puis optimiser des ligands de deux classes de métalloenzymes que sont les aminopeptidases du réticulum endoplasmiques (ERAP) et l’élastase LasB de la bactérie Pseudomonas aeruginosa. Les ERAPs (1 et 2) participent au processus de maturation des antigènes. Ces enzymes clivent les précurseurs peptidiques en peptides antigéniques matures afin que ceux-ci disposent d’une taille optimale pour leur complexation au complexe majeur d’histocompatibilité de classe I et ainsi initient ou non la réponse immunitaire adaptative. Les niveaux d’expression de ces protéases ainsi que des polymorphismes d’un seul nucléotide ont été associé au développement de cancers et de maladies auto-immunes. Ainsi, la modulation de ces enzymes permettrait de lutter contre les pathologies associées au système immunitaire. P. aeruginosa est une bactérie Gram negative dotée d’une virulence et d’une résistance aux antimicrobiens remarquable. Aujourd’hui, la résistance aux antibiotiques représente un enjeu de santé publique majeur et il y a un besoin urgent en nouvelles thérapeutiques. Afin de satisfaire ce besoin, de nouvelles stratégies sont apparues comme celle consistant à cibler la virulence des bactéries afin de « désarmer » celles-ci. LasB représente une cible thérapeutique de choix de par sa localisation extracellulaire et ses implications physiopathologiques (colonisation, invasion, évasion à la réponse immunitaire, formation de biofilm, etc.). Bien qu'il y ait un besoin médical évident non satisfait dans ces deux aires thérapeutiques, aucun modulateur des ERAPs ni de LasB n'a atteint le marché. Ainsi, l’utilisation de la stratégie KTGS suivie de phases d’optimisation nous ont permis d’identifier et optimiser de nouvelles familles de ligands de ces enzymes. Ces composés peuvent être considérés comme des leads prometteurs puisqu’ils présentent des affinités nanomolaires pour leurs cibles respectives, des profils de sélectivité et de toxicité ainsi que des propriétés physicochimiques remarquables
Target-guided synthesis of protein ligands is an innovative strategy to discover bioactive compounds. In particular, the Kinetic Target-Guided Synthesis (KTGS) and the Dynamic Combinatorial Chemistry (DCC) have allowed, in recent years, the discovery of novel ligands for poorly explored therapeutic targets, which has enabled drug-discovery projects. This thesis project aims at using KTGS to discover and optimize ligands for two classes of metalloenzymes, namely endoplasmic reticulum aminopeptidases (ERAPs) and elastase LasB from the bacterium Pseudomonas aeruginosa. ERAPs (1 and 2) are involved in the process of antigen maturation. These enzymes cleave peptide precursors into mature antigenic peptides so that they have an optimal size for their complexation to the major histocompatibility complex of class I and thus initiate or not the adaptive immune response. The expression levels of these proteases as well as single nucleotide polymorphisms have been associated with the development of cancers and autoimmune diseases. Thus, the modulation of these enzymes would allow to fight against pathologies associated with the immune system. P. aeruginosa is a Gram-negative bacterium with remarkable virulence and antimicrobial resistance. Today, antibiotic resistance represents a major public health issue and there is an urgent need for new therapeutics. In order to meet this need, new strategies have emerged such as targeting the virulence of bacteria to "disarm" them. LasB represents a therapeutic target of choice due to its extracellular localization and its physiopathological implications (colonization, invasion, evasion of immune response, biofilm formation, etc.). Although there is a clear unmet medical need in these two therapeutic areas, no modulator of ERAPs or LasB has reached the market. Thus, the use of the KTGS strategy followed by optimization phases allowed us to identify and optimize new families of ligands for these enzymes. These compounds can be considered as promising lead compounds since they present nanomolar affinities for their respective targets, selectivity and toxicity profiles as well as remarkable physicochemical properties

Carbonic anhydrases (CAs) are zinc metalloenzymes that catalyze the reversible hydration of carbon dioxide to bicarbonate and a proton. The zinc cation in the active site of these metalloenzymes is the suggested target for small molecule inhibitors to block the endogenous CA catalysed reaction. Almost all reported CA inhibitors consist of zinc binding group (ZBG) among which the, primary sulfonamide group (-SO2NH2) is the most prominent example. Primary sulfamate (-OSO2NH2) and primary sulfamide (-NH-SO2NH2) groups also serve as zinc binding groups in CA inhibitors, however they are less represented than the primary sulfonamide group. Natural products (NPs) have proven an invaluable source of compounds for drug discovery. NPs containing primary sulfonamide (-SO2NH2) and primary sulfamate (-OSO2NH2) group are rare, while there are no NP primary sulfamides. A literature search of the Dictionary of Natural Products (DNP) database revealed only two primary sulfonamide and five primary sulfamate compounds. This project will focus on the tractable synthesis of these rare natural products, biological evaluation against CA and protein X-ray crystal structures in complex with medicinally important isozymes of CA.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Natural Sciences
Science, Environment, Engineering and Technology
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La résistance aux antibiotiques est une menace sérieuse pour la santé publique. En effet, si on ne change pas rapidement notre consommation excessive d'antibiotiques, la situation actuelle va se dégrader jusqu'à basculer dans une ère dite "post-antibiotique", dans laquelle plus aucun antibiotique ne sera efficace contre les infections microbiennes. Bien que ce phénomène de résistance apparaît naturellement, l'utilisation abusive d'antibiotiques accélère le processus. De plus, la présence de pathogènes multi-résistants neutralise l'effet des traitements existants et dans le cas de chirurgies courantes (césariennes, transplantations d'organe...), la situation peut rapidement s'aggraver voire devenir mortelle. C'est pourquoi des directives, émanant des autorités sanitaires, doivent être mises en place afin de contrôler l'utilisation des médicaments, et ce, à tous les niveaux de la société, des individus au secteur agricole en passant par les professionnels de santé et les industries pharmaceutiques. Le monde de la recherche scientifique, quant à elle, doit trouver des nouvelles stratégies pour enrayer la propagation de la résistance. Dans ce contexte, cette thèse a pour objectif le développement d'une méthode de prédiction, par calculs d'énergie libre, des mutations de β-lactamases favorables à l'hydrolyse des β-lactames. Ces travaux méthodologiques ont donc conduit au développement : (1) de nouveaux paramètres pour les enzymes à zinc, implémentés dans le champ de force OPLS-AA et validés par des simulations de dynamique moléculaire sur un panel de métalloenzymes représentatives, (2) d'un protocole de paramétrisation de ligands covalents pour étudier le comportement de certains β-lactames dans CMY-136, une nouvelle β-lactamase caractérisée au laboratoire, et (3) d'un protocole de calcul d'énergie libre évalué au moyen de compétitions internationales de prédiction. Ce dernier a ensuite été utilisé pour tenter d'expliquer pourquoi la carbamylation de la sérine catalytique n'a pas lieu dans certaines oxacillinases. Au travers de ces travaux, nous avons pu améliorer significativement notre approche computationnelle et désormais tout est en place pour une exploration exhaustive des mutations possibles dans les β-lactamases
Antibiotic resistance is a global concern threatening worldwide health. Indeed, if we don't change our overconsumption of antibiotics, the current situation could worsen until a "post-antibiotic" era in which existing treatment would be ineffective against microbial infections. Despite the natural occurrence of antibiotic resistance, the misuse of antibiotics is speeding up the process. Furthermore, presence of multi-resistant pathogens negates the effect of modern treatments and usual surgeries (caesarean sections, organ transplantations...) might be riskier in the future, or even lethal. That's why, common guidelines have to be edicted by health authorities in order to control antibiotic use at every level of society, from individuals to healthcare industry including health professionals and agriculture sector. As for scientific research, new strategies have to be considered in order to limit the spread of antibiotic resistance. In that context, the presented thesis aimed at developing a protocol to predict, by free energy calculations, β-lactamase mutations which could promote the hydolysis of β-lactams antibiotics. In order to achieve that, we developed several methodological approaches including: (1) new parameters for zinc enzymes implemented in OPLS-AA force field and thereafter validated using molecular dynamics simulations of representative zinc-containing metalloenzymes, (2) a protocol to parameterize covalent ligands in order to analyze the dynamical behavior of some β-lactams in CMY-136, a novel β-lactamase recently characterized in our laboratory, and (3) a pmx-based free energy protocol. The latter was also assessed through several international blinded prediction challenges, and finally used to find out why carbamylation of the catalytic serine is not observed in certain OXA enzymes. Throughout this work, we made significant improvements in our protocol, and now everything is in place for an exhaustive prediction of possible mutations in β-lactamases

The principal methylglyoxal (MG)-detoxifying system in most living organisms is the two metalloenzyme Glyoxalase system. Glyoxalase I (GlxI) initially converts the non-enzymatically formed MG-GSH hemithioacetal to the thioester S,D-lactoylglutathione. The hydrolase, Glyoxalase II(GlxII) regenerates GSH and liberates the product D-lactate. Ni2+/Co2+- and Zn2+-activated GlxI enzymes exist in nature. The Ni2+/Co2+-activated GlxI are not active as Zn2+-holoenzymes in spite of the structural similarities to the Zn2+-dependent enzymes. The Zn2+-GlxI enzymes have been investigated heavily relative to the Ni2+/Co2+-activated enzymes, which have been isolated more recently. As part of this study the three GlxI homologs isolated from Pseudomonas aeruginosa were characterized. The homologous genes encode GlxI enzymes of both metal activation type. The Zn2+-activated P. aeruginosa GlxI is difficult to de-metallate compared to the Ni2+/Co2+-activated enzymesreflecting a difference in metal-binding/insertion between the two types of GlxI. The E. coli GlxII was isolated and characterized to determine whether Ni2+/Co2+-activation is a characteristic of the Glx system as a whole in this organism. Inductively coupled plasma mass spectrometry on purified E. coli GlxII confirms that the active protein is a binuclear Zn2+-metalloenzyme. The results to date indicate a detectable isotope effect for the Cd2+-holoenzyme but not the Ni2+-reconstituted enzyme. Chemical crosslinking experiments indicate that the SlyD Ni2+ metallochaperone does not form a complex with E.coli GlxI. This indicates that the E. coli active site is not metallated in vivo by this accessory protein. The principal biophysical experiment in this project was determining of Ni2+-binding stoichiometry for E. coli GlxI by 1H-15N heteronuclear single quantum coherence (HSQC) NMR. The GlxI dimer reorganization ceases when the metal:dimer stoichiometry reaches 0.5 during apoenzyme titration. This finding supports previous studies that indicate half-of-the-sites metal binding in this enzyme.

Amyotrophic lateral sclerosis (ALS) is a devastating disease characterized by the progressive degeneration of motor neurons. Dominantly-inherited mutations to the antioxidant enzyme Cu,Zn superoxide dismutase (SOD1) cause 3-6% of all ALS cases. The complete mechanism behind the toxicity of mutant SOD1 remains unclear, although significant evidence points to aberrant or incomplete metal-binding having a role in a toxic gain-of-function. However, the relevance of the metal-binding of SOD1 to mutant-SOD1-linked ALS remains controversial. Direct assessments of protein metal-binding from transgenic, SOD1-overexpressing rodent models of the disease are difficult to acquire due to the non-covalent nature of the interaction. The relatively small amount of disease-afflicted spinal cord tissue in which the motor neurons reside compounds the difficulty of measuring the protein metal binding of SOD1 from transgenic mice. This dissertation addresses the metals bound to SOD1 throughout the disease course in transgenic mice using a novel mass spectrometry assay. The methodology developed here offers the first detailed examination of partially unfolded intermediates of SOD1 present in the spinal cord of pre-symptomatic, symptomatic, and end-stage transgenic mice overexpressing the ALS-associated SOD1 mutation G93A (glycine mutated to alanine at position 93). These results were compared to age-matched transgenic mice expressing wild-type SOD1 that do not develop ALS symptoms. To extract SOD1 from relevant spinal cord tissue, a 300 ��m necropsy punch was used to remove a small piece of tissue from the ventral or dorsal gray matter of a 1 mm-thick slice of spinal cord. Physiological salts that interfere with electrospray mass spectrometry were removed by binding the proteins to a C4 Ziptip��, a pipette tip containing hydrophobic, reversed-phase packing material. Washing the Ziptip-bound proteins with water eliminated interfering salts. Bound proteins could then be eluted into a mass spectrometer with low concentrations of acetonitrile plus formic acid. Electrospray ionization conditions were determined that could keep both copper and zinc bound to SOD1. Using a high-resolution Fourier transform-ion cyclotron resonance mass spectrometer, we used the assay to collect isotopically-resolved protein mass data. Theoretical protein isotope distributions were calculated from the empirical formulas of SOD1 and matched to the experimental data with a least squares fitting algorithm to determine the multiple intermediates of SOD1 present. Spinal cord tissue, wild-type in particular, was notable for containing significantly more one-metal SOD1 than any other tissue, despite having 3-fold less SOD1 than liver. We quantitatively compared the levels of soluble, partially unfolded intermediates of SOD1 from wild-type and G93A SOD1 spinal cords. Wild-type mouse spinal cord contained significantly more of all of the partially unfolded intermediates copper-deficient SOD1, disulfide reduced SOD1, and apo SOD1. The amount of zinc-containing SOD1 was exceptionally high in wild-type mice, comprising 60% of the total SOD1 in wild-type spinal cord. The larger amounts of these SOD1 intermediates in wild-type transgenic mice indicate that they are not directly responsible for toxicity in vivo. However, copper-containing, zinc-deficient SOD1 was the one species found in higher concentrations in G93A SOD1 spinal cord. The concentration was on average 0.6-0.8 ��M in G93A spinal cord, compared to 0.1-0.3 ��M zinc-deficient SOD1 found in the wild-type mouse spinal cord. A concentration above 0.5 ��M zinc-deficient SOD1 was sufficient to induce motor neuron death in vitro. These results suggest that copper-containing, zinc-deficient SOD1 could be the toxic species responsible for motor neuron death in ALS.
Graduation date: 2013

Metallohydrolases with dinuclear-zinc active sites perform many important biological hydrolytic reactions on a variety of substrates. In this regard, metallo-β-lactamases (mβ1, class B) represent a unique subset of zine hydrolases that hydrolyze the β-lactam ring in several antibiotics. The antibiotic resistance that results from this hydrolysis is becoming an increased threat for the clinical community. These metalloenzymes can hydrolyze a wide range of β-lactam substrates, such as cephamycins and imipenem that are generally resistant t the serine-containing β-lactamases. Therefore, the clinical application of the entire range of antibiotics is severely compromised in bacteria that produce mβls. Due to the lack of information on the mechanism of mβls, to-date, no clinically known inhibitors is there for mβls. In this present study, we synthesized several mono and dizinc complexes as models for the mβls and investigated the differences in their hydrolytic properties. This study supports the assumption that the second zinc in the dinuclear enzymes does not directly involve in the catalysis, but may orient the substrates for hydrolysis and the basic amino acid residues such as Asp and His may activate the zinc-bound water molecules, fulfilling the role of the second zinc in the mononuclear enzymes. The effect of various side chains on the hydrolysis of some commonly used cephalosporin antibiotics by mβl from B.cereus is described. It is shown that the cephalosporins having heterocyclic thiol side chains are more resistance to mβl-mediated hydrolysis than the antibiotics that do not have such side chains. This is partly due to the inhibition of enzyme activity by the thiol moieties eliminated during the hydrolysis. It is also observed that the heterocyclic side chains in pure form inhibit the lactamase activity of mβl as well as its synthetic mimics. The mode of binding of these heterocyclic side chains to the zinc has been analyzed from the crystal structure of the tetranuclear zinc complexes. The theoretical studies suggest that the eliminated heterocyclic thiols undergo a rapid tautomerism to produce the corresponding thiones. These thiones are found to irreversibly inhibit the LPO-catalyzed iodination reaction. The reaction of various thiones with I2 leads to the formation of thione-iodine complexes similar to that of the most commonly used antithyroid drug methimazole(MMI). These observations suggest that some of the latest generation of antibiotics may show negative effects on thyroid gland upon hydrolysis. Synthetic organophosphorus compounds have been used extensively as pesticides and petroleum additives. These compounds are very toxic to mammals and their widespread use in agriculture leads to serious environmental problems. Therfore, degradation of organophosphorus trimesters and remediation of associated contaminated sites are of worldwide concern. In this regards, the bacterial phsophotriesterase (PTE) enzyme plays an important role in degrading a wide range of organophosphorus esters and the active side of PTE has been shown to be very similar to that of mβl. This identification prompted us to check the hydrolysis of phosphotriesters by the mβl and its mimics. It has been observed that the dinuclear zine(II) complexes that do not allow a strong binding of phosphodiestes would be a better PTE mimics.