2-Cys Prx compõem um grupo de enzimas antioxidantes homodiméricas que atuam na decomposição de hidroperóxidos utilizando uma cisteína reativa (cisteína peroxidásica - CysP). A alta reatividade da CysP é alcançada com o envolvimento de dois aminoácidos vicinais à CysP: uma treonina e uma arginina, que constituem a tríade catalítica. Após a decomposição do hidroperóxido, a CysP forma um dissulfeto intermolecular com um segundo resíduo de cisteína (cisteína de resolução - CysR), o qual é reduzido pela tiorredoxina (Trx). Durante o ciclo redox, estas enzimas sofrem alterações estruturais, mas os mecanismos envolvidos neste processo eram pouco compreendidos. Neste trabalho foi obtida a estrutura cristalográfica de Tsa1 de Saccharomyces cerevisiae, uma 2-Cys Prx. Através de abordagens envolvendo bioquímica e biologia molecular, foi verificada a importância de aminoácidos envolvidos na reatividade e em transições da estrutura terciária e quaternária. Por fim, foram realizados esforços para a determinação da estrutura cristalográfica de mutantes obtidos neste trabalho. / 2-Cys Prx constitute a group of homodimeric antioxidant enzymes that act in the decomposition of hydroperoxides using a reactive cysteine (peroxidase cysteine - CysP). The high reactivity of the CysP is achieved by the participation of two vicinal amino acids: a threonine and an arginine, which constitute the catalytic triad. After the decomposition of hydroperoxide, the CysP forms an intermolecular disulfide with a second cysteine residue (resolving cysteine - CysR), which is reduced by the thioredoxin (Trx). During the redox cycle, these enzymes undergo to changes in the structure, but the molecular mechanisms involved in this process were poorly understood. In this study we have obtained the crystallographic structure of the 2-Cys Prx enzyme Tsa1 from Saccharomyces cerevisiae. By means of biochemical and molecular biology approaches, the importance of amino acids involved in reactivity and structural transitions were determined. Finally, efforts have been performed to the determination of the crystallographic structures of mutant proteins obtained in this study.

2-Cys Prx compõem um grupo de enzimas antioxidantes homodiméricas que atuam na decomposição de hidroperóxidos utilizando uma cisteína reativa (cisteína peroxidásica - CysP). A alta reatividade da CysP é alcançada com o envolvimento de dois aminoácidos vicinais à CysP: uma treonina e uma arginina, que constituem a tríade catalítica. Após a decomposição do hidroperóxido, a CysP forma um dissulfeto intermolecular com um segundo resíduo de cisteína (cisteína de resolução - CysR), o qual é reduzido pela tiorredoxina (Trx). Durante o ciclo redox, estas enzimas sofrem alterações estruturais, mas os mecanismos envolvidos neste processo eram pouco compreendidos. Neste trabalho foi obtida a estrutura cristalográfica de Tsa1 de Saccharomyces cerevisiae, uma 2-Cys Prx. Através de abordagens envolvendo bioquímica e biologia molecular, foi verificada a importância de aminoácidos envolvidos na reatividade e em transições da estrutura terciária e quaternária. Por fim, foram realizados esforços para a determinação da estrutura cristalográfica de mutantes obtidos neste trabalho. / 2-Cys Prx constitute a group of homodimeric antioxidant enzymes that act in the decomposition of hydroperoxides using a reactive cysteine (peroxidase cysteine - CysP). The high reactivity of the CysP is achieved by the participation of two vicinal amino acids: a threonine and an arginine, which constitute the catalytic triad. After the decomposition of hydroperoxide, the CysP forms an intermolecular disulfide with a second cysteine residue (resolving cysteine - CysR), which is reduced by the thioredoxin (Trx). During the redox cycle, these enzymes undergo to changes in the structure, but the molecular mechanisms involved in this process were poorly understood. In this study we have obtained the crystallographic structure of the 2-Cys Prx enzyme Tsa1 from Saccharomyces cerevisiae. By means of biochemical and molecular biology approaches, the importance of amino acids involved in reactivity and structural transitions were determined. Finally, efforts have been performed to the determination of the crystallographic structures of mutant proteins obtained in this study.

L. pneumophila, der Erreger der Legionärskrankheit, kodiert für eine Vielzahl lipolytischer Enzyme. Bis zu 17 verschiedenen Proteinen kann aufgrund von Sequenzhomologien oder experimenteller Analyse phospholipolytische Eigenschaft zugeschrieben werden. Neben sekretierten Formen wird eine besonders aktive zell-assoziierte Variante exprimiert, die Phospholipase A/Lysophospholipase A PlaB. Wie bereits gezeigt werden konnte, kodiert das plaB Gen für die hauptsächliche membranständige Phospholipase A von L. pneumophila mit Enzymaktivitäten, die die Aktivität sekretierter Proteine um das 100-fache übersteigen. Da PlaB zu keiner der bisher beschriebenen Phospholipasen Homologien aufweist, wurden in dieser Arbeit durch gezielte Mutagenese die katalytisch wichtigen Aminosäuren identifiziert. Dies ergab, dass PlaB zwar eine für Lipasen und Proteasen typische katalytische Triade aus Serin, Asparat und Histidin ausbildet, die umliegenden Motive sich aber deutlich von bisher beschriebenen Enzymklassen unterscheiden. Somit stellt PlaB das erste näher charakterisierte Mitglied einer neuen Familie phospholipolytischer Enzyme dar. Im Weiteren konnten für die Substratspezifität wichtige Aminosäurereste identifiziert werden. Dabei stellte sich heraus, dass die Fähigkeit zur Hydrolyse von cholinkettentragenden Substraten besonders suszeptibel gegenüber Mutationen war. Da im Vergleich zu nicht-pneumophila Stämmen, wie z. B. L. spiritensis, nur L. pneumophila in der Lage war, diese Lipide in hohem Maße umzusetzen, kann die Eigenschaft von PlaB, Phosphatidylcholin (PC) zu hydrolysieren, einen Virulenzvorteil für L. pneumophila bedeuten. Die Hypothese konnte durch Hämolyse-experimente bestärkt werden. Hier zeigten sich Mutanten mit reduziertem Potential zur Hydrolyse von PC weniger zytotoxisch gegenüber humanen Erythrozyten. Das zell-zerstörende Potential von PlaB könnte somit eine enorme Auswirkung auf die Virulenzeigenschaften von L. pneumophila haben. Wie in der vorliegenden Arbeit untersucht, bestätigten in vitro Experimente, dass PlaB die hauptsächliche Aktivität während einer Makrophageninfektion darstellt, die Deletion des Gens aber keine Auswirkungen auf das Replikationspotential der Bakterien hat. Ganz im Gegenteil dazu waren plaB Insertionsmutanten bei der Infektion von Meerschweinchen in ihrer Vermehrungsfähigkeit in der Lunge als auch in der Verbreitung der Erreger zur Milz der Tiere reduziert. Um den Grund des Defektes näher zu erörtern, wurde in einem Screen auf 40 verschiedene Entzündungsmediatoren die Sekretion von IL-8, MCP-1, RANTES und TIMP-2 als PlaB-abhängig identifiziert. Somit repräsentiert die zell-assoziierte Phospholipase A, PlaB, von L. pneumophila eine neue Klasse lipolytischer Enzyme und kann durch Hydrolyse eines breiten Substratspektrums, insbesondere durch Hydrolyse von PC, die Vermehrung und Verbreitung des Erregers im Wirtsorganismus unterstützen. / L. pneumophila, the causative agent of Legionnaires’ disease (LD) expresses numerous lipolytic enzymes. According to sequence homology or determined lipolytic activities, up to 17 open reading frames of the L. pneumophila genome may encode functional phospholipases. In addition to secreted and/or injected lipolytic enzymes, it was shown that the pathogen expresses a highly active and membrane-bound phospholipase A/lysophospholipase A with hemolytic activity, designated PlaB. As PlaB does not belong to any established bacterial or eukaryotic protein family of lipolytic enzymes nor does it show sequence homology to conserved motifs harboring the catalytically important amino acids, we analyzed putative catalytic centers using site-directed mutagenesis. This study shows that PlaB exhibits a catalytic triad of serine, aspartate and histidine residues, most commonly found within lipolytic and proteolytic enzyme families. However, surrounding motifs differ significantly from described ones. Thus, PlaB is the first representative of a new class of lipolytic enzymes. In addition, we described amino acids important for substrate specificity, revealing that the ability to hydrolyze phosphatidylcholine (PC) is severely susceptible to various mutations. Since PlaB of non-pneumophila strains, such as L. spiritensis, express comparable activities against glycerol-containing lipids, but are reduced in their hydrolytic potential to cleave choline-containing substrates, PC-targeting activity could be an important contribution to the pathogenicity of L. pneumophila, the most common cause of LD. The hypothesis was underlined by reduced hemolytic potential of L. spiritensis PlaB and PC-hydrolysis impaired mutants of L. pneumophila PlaB and is in accordance with PC being the major lipid in the outer leaflet of eukaryotic membranes. The cell destructive properties of PlaB may enhance bacterial pathogenicity in multiple ways. As depicted within this study, PlaB represents the major lipolytic activity present throughout host cell infections; however, gene deletion mutants retained their ability to multiply within several host cell infection systems. On the contrary, the plaB mutant strain was inhibited in replicating in the lung and disseminating to other organs in a guinea pig infection model. To elucidate the impact of PlaB on Legionella virulence we investigated 40 inflammatory factors secreted by lung epithelial cells upon Legionella infection and observed that IL-8, MCP-1, RANTES and TIMP-2 are released in a PlaB-dependent manner. Thus, PlaB represents a new family of lipolytic enzymes which could, according to the lipolytic profile and especially the ability to hydrolyse PC, contribute to replication and dissemination properties of a pathogen within a host cell, e.g. amoeba, or even more complex organisms such as guinea pigs or humans.