Inibidores tipo Kunitz são proteínas de aproximadamente 20 kDa, que geralmente possuem de dois a quatro resíduos de cisteína, formando uma ou duas pontes dissulfeto, sendo responsáveis por inibir uma ou diversas serina proteinases com grande especificidade. Eles são importantes tanto por atuarem em situações de defesa na planta como por estarem envolvidos na inibição de diversas proteinases, como aquelas presentes na cascata de coagulação sanguínea, no processo inflamatório ou e até mesmo atuarem na supressão de tumores. Este trabalho teve como alvo o estudo de um inibidor tipo Kunitz encontrado em sementes de Bauhinia bauhinioides (Martius) Macbr., denominado BbKI (Bauhinia bauhinioides Kallikrein Inhibitor). Um fragmento gênico codificando a seqüência primária madura de BbKI foi amplificado por RT-PCR e clonado no vetor pGEM-T. Através da técnica de RACE (3 e 5) foi possível constatar que a proteína é sintetizada inicialmente como um prepropeptídeo com a seguinte estrutura: peptídeo sinal (19 resíduos de aminoácidos), proteína madura (164 resíduos), e peptídeo C-terminal (10 resíduos). A presença do peptídeo sinal demonstra que a proteína segue uma rota de síntese via retículo endoplasmático e sugere que este inibidor possa seguir para outro compartimento celular, sinalizado pelo peptídeo C-terminal. Para avaliar se este peptídeo não seria um mero inibidor da atividade biológica, foram feitas duas subclonagens em vetores do sistema pET: uma com o fragmento gênico codificador para BbKI madura, e outra adicionando a seqüência codificante para a porção C-terminal na proteína madura. As proteínas recombinantes foram expressas em células de E. coli BL21(DE3), as quais foram purificadas através de cromatografia de afinidade (Ni-NTA) e filtração em gel, apresentando a massa molecular esperada de 20 kDa. Testes de atividade com tripsina mostraram que ambas as proteínas são biologicamente ativas, embora com diferentes constantes de inibição. Estudos de Dicroísmo Circular revelaram estruturas secundárias similares para ambas as proteínas. Quando analisado por espectroscopia de fluorescência, o inibidor maduro mostrou-se estável numa ampla faixa de pH. A proteína madura recombinante foi ainda cristalizada e o cristal foi difratado por raios X até a resolução máxima de 1,9 A, permitindo a resolução e o refinamento de sua estrutura. A análise da estrutura revelou que o inibidor apresenta um enovelamento -trefoil que é típico nos inibidores tipo Kunitz previamente estudados. Sua estrutura terciária mostrou que no centro ativo o loop inibitório está exposto ao solvente e que os únicos W e C ficam escondidos no interior da proteína, rodeados por resíduos hidrofóbicos / Kunitz-type inhibitors are proteins of about 20 kDa that usually contain from 2 to 4 cystein residues used to form one or two dissulfide bridges. They are responsible for inhibiting one or severa1 serine proteinases with large specificity. Kunitz inhibitors are important both for playing defense roles in plants and also for being involved in the inhibition of many proteinases, like those present in the blood coagulation cascade, in inflammatory processes, or even for suppressing tumors. The aim of this work was to study a Kunitz-type inhibitor from Bauhinia bauhinioides (Martius) Macbr. seeds, denominated BbKI (Bauhinia buhinioides Kallikrein Inhibitor). A gene fragment codifying the mature primary sequence of BbKI was amplified by RT-PCR and was cloned in the pGEM-T vector. Using the RACE (3 and 5) technique we could verify that this protein is synthesized as a prepropeptide with the following structure: signal peptide (19 amino acid residues), mature protein (164 residues) and C-terminal peptide (10 residues). The presence of the peptide signal shows this protein follows a synthesis pathway via endoplasmatic reticulum and suggests the inhibitor can move to another cell compartment guided by the C-terminal peptide. To evaluate if this peptide was just an inhibitor of the biological activity, we performed two subclonings in the system pET vectors: one using the gene fragment codifying to mature BbKI, and another adding the codifying sequence for the C-terminal peptide to the mature protein. The recombinant proteins were expressed in E. coli BL21(DE3) cells which were purified by affinity chromatography (Ni-NTA) and gel filtration thus presenting the expected molecular mass of 20kDa. Activity assays with trypsin showed that both of proteins are biologically active, although they presented different inhibition constants. Circular Dichroism studies revealed that both proteins have similar secondary structures. When analyzed by fluorescence spectroscopy the mature inhibitor was stable in a wide pH range. The mature recombinant protein was latter crystallized and the crystal was diffracted by X-ray at 1.9A resolution, allowing the resolution and refinement of its structure. The analysis of this structure revealed the inhibitor presents a -trefoil fold, which is typical in the Kunitz-type inhibitors previously studied. Its tertiary structure showed that in the active site the inhibitory loop is exposed to solvent, and that the W and the C are buried into the protein surrounded by hydrophobic residues.

Inibidores tipo Kunitz são proteínas de aproximadamente 20 kDa, que geralmente possuem de dois a quatro resíduos de cisteína, formando uma ou duas pontes dissulfeto, sendo responsáveis por inibir uma ou diversas serina proteinases com grande especificidade. Eles são importantes tanto por atuarem em situações de defesa na planta como por estarem envolvidos na inibição de diversas proteinases, como aquelas presentes na cascata de coagulação sanguínea, no processo inflamatório ou e até mesmo atuarem na supressão de tumores. Este trabalho teve como alvo o estudo de um inibidor tipo Kunitz encontrado em sementes de Bauhinia bauhinioides (Martius) Macbr., denominado BbKI (Bauhinia bauhinioides Kallikrein Inhibitor). Um fragmento gênico codificando a seqüência primária madura de BbKI foi amplificado por RT-PCR e clonado no vetor pGEM-T. Através da técnica de RACE (3 e 5) foi possível constatar que a proteína é sintetizada inicialmente como um prepropeptídeo com a seguinte estrutura: peptídeo sinal (19 resíduos de aminoácidos), proteína madura (164 resíduos), e peptídeo C-terminal (10 resíduos). A presença do peptídeo sinal demonstra que a proteína segue uma rota de síntese via retículo endoplasmático e sugere que este inibidor possa seguir para outro compartimento celular, sinalizado pelo peptídeo C-terminal. Para avaliar se este peptídeo não seria um mero inibidor da atividade biológica, foram feitas duas subclonagens em vetores do sistema pET: uma com o fragmento gênico codificador para BbKI madura, e outra adicionando a seqüência codificante para a porção C-terminal na proteína madura. As proteínas recombinantes foram expressas em células de E. coli BL21(DE3), as quais foram purificadas através de cromatografia de afinidade (Ni-NTA) e filtração em gel, apresentando a massa molecular esperada de 20 kDa. Testes de atividade com tripsina mostraram que ambas as proteínas são biologicamente ativas, embora com diferentes constantes de inibição. Estudos de Dicroísmo Circular revelaram estruturas secundárias similares para ambas as proteínas. Quando analisado por espectroscopia de fluorescência, o inibidor maduro mostrou-se estável numa ampla faixa de pH. A proteína madura recombinante foi ainda cristalizada e o cristal foi difratado por raios X até a resolução máxima de 1,9 A, permitindo a resolução e o refinamento de sua estrutura. A análise da estrutura revelou que o inibidor apresenta um enovelamento -trefoil que é típico nos inibidores tipo Kunitz previamente estudados. Sua estrutura terciária mostrou que no centro ativo o loop inibitório está exposto ao solvente e que os únicos W e C ficam escondidos no interior da proteína, rodeados por resíduos hidrofóbicos / Kunitz-type inhibitors are proteins of about 20 kDa that usually contain from 2 to 4 cystein residues used to form one or two dissulfide bridges. They are responsible for inhibiting one or severa1 serine proteinases with large specificity. Kunitz inhibitors are important both for playing defense roles in plants and also for being involved in the inhibition of many proteinases, like those present in the blood coagulation cascade, in inflammatory processes, or even for suppressing tumors. The aim of this work was to study a Kunitz-type inhibitor from Bauhinia bauhinioides (Martius) Macbr. seeds, denominated BbKI (Bauhinia buhinioides Kallikrein Inhibitor). A gene fragment codifying the mature primary sequence of BbKI was amplified by RT-PCR and was cloned in the pGEM-T vector. Using the RACE (3 and 5) technique we could verify that this protein is synthesized as a prepropeptide with the following structure: signal peptide (19 amino acid residues), mature protein (164 residues) and C-terminal peptide (10 residues). The presence of the peptide signal shows this protein follows a synthesis pathway via endoplasmatic reticulum and suggests the inhibitor can move to another cell compartment guided by the C-terminal peptide. To evaluate if this peptide was just an inhibitor of the biological activity, we performed two subclonings in the system pET vectors: one using the gene fragment codifying to mature BbKI, and another adding the codifying sequence for the C-terminal peptide to the mature protein. The recombinant proteins were expressed in E. coli BL21(DE3) cells which were purified by affinity chromatography (Ni-NTA) and gel filtration thus presenting the expected molecular mass of 20kDa. Activity assays with trypsin showed that both of proteins are biologically active, although they presented different inhibition constants. Circular Dichroism studies revealed that both proteins have similar secondary structures. When analyzed by fluorescence spectroscopy the mature inhibitor was stable in a wide pH range. The mature recombinant protein was latter crystallized and the crystal was diffracted by X-ray at 1.9A resolution, allowing the resolution and refinement of its structure. The analysis of this structure revealed the inhibitor presents a -trefoil fold, which is typical in the Kunitz-type inhibitors previously studied. Its tertiary structure showed that in the active site the inhibitory loop is exposed to solvent, and that the W and the C are buried into the protein surrounded by hydrophobic residues.

Die lösliche, NAD+-reduzierende Hydrogenase (SH) aus Ralstonia eutropha H16 ist eine Pyridinnukleotid-abhängige Hydrogenase. Das heißt, der Umsatz von H2 im Hydrogenasemodul des Enzyms ist an die Reduktion von NAD(P)+ im NAD(P)H:Akzeptor-Oxidoreduktasemodul gekoppelt. Die SH ist Vertreter des Subtyps, der auch in Gegenwart von O2 katalytisch aktiv ist. Dies wird ermöglicht durch eine reduktive Entfernung von O2, die nach dem aktuellen Modell abhängig ist vom rückläufigen e--Transport vom NADH:Akzeptor-Oxidoreduktasemodul zum aktiven [NiFe]-Zentrum in der großen Hydrogenaseuntereinheit. Der Einfluss des FeS-Clusters in der kleinen Hydrogenaseuntereinheit HoxY auf diesen Prozess wurde hier untersucht. Dabei konnte gezeigt werden, dass die vier hochkonservierten Cysteine C41, C44, C113 und C179 in HoxY an der Koordination des FeS-Zentrums beteiligt sind. Außerdem wurde das nahegelegene Cystein C39 als relevant für die Sauerstofftoleranz identifiziert. Weiterhin wurde gezeigt, dass das Tryptophan W42 aus HoxY essentiell für die Hydrogenaseaktivität der SH ist. Damit bestätigt sich, dass die Kinetik des rückläufigen e--Transports durch die Aminosäureumgebung des FeS-Clusters in HoxY beeinflusst ist. Ferner wurden in dieser Arbeit Ansätze zum Einsatz der SH aus R. eutropha in einer H2-produzierenden cyanobakteriellen Designzelle weiterverfolgt. Dazu wurden Hybridsysteme aus SH und cyanobakteriellem Photosystem I in vitro hinsichtlich ihrer Fähigkeit zur lichtgetriebenen H2-Produktion untersucht. Außerdem wurde an einem heterologen Expressionssystem der SH für Cyanobakterien gearbeitet. Weiterhin wurde die SH aus Rhodococcus opacus MR11 als komplementäres Modellsystem für O2-tolerante Pyridinnukleotid-abhängige Hydrogenasen etabliert. Dieser zur SH aus R. eutropha homologe Komplex hatte in früheren Arbeiten Vorteile für spektroskopische Studien offenbart und wurde hier erstmals im direkten Vergleich zur SH aus R. eutropha biochemisch und spektroskopisch charakterisiert. / The soluble, NAD+-reducing hydrogenase (SH) from Ralstonia eutropha H16 is a pyridine nucleotide-dependent hydrogenase. In these types of enzymes the conversion of H2 in the hydrogenase module of the complex is coupled to the reduction of NAD(P)+ in the NAD(P)H:acceptor oxidoreductase module. The SH belongs to a subtype that is catalytically active also in the presence of O2. This O2 tolerance is enabled by a reductive removal of O2, which according to the current model depends on a reverse e- flow from the NADH:acceptor oxidoreductase module to the active [NiFe] site in the large hydrogenase subunit. The impact of the FeS cluster in the small hydrogenase subunit HoxY on this process was analyzed in this study. Thereby it was shown that the four highly conserved cysteines C41, C44, C113 and C179 in HoxY are involved in the coordination of the FeS center. Further the nearby cysteine C39 was identified to be relevant for the O2 tolerance of the SH. Additionally we found the tryptophan W42 to be essential for the hydrogenase activity of the SH. Thus it was confirmed that the kinetic of the reverse e- transport is affected by the amino acid environment of the FeS cluster in HoxY. In addition, approaches for using the SH from R. eutropha in H2 producing cyanobacterial design cells were pursued. On one hand hybrid systems consisting of the SH and cyanobacterial photosystem I were analyzed in vitro for their capacity to produce H2 in a light dependent manner. On the other hand work on a heterologous expression system of the SH for Cyanobacteria was continued. Furthermore the SH from Rhodococcus opacus MR11 was established as complementary model system for O2-tolerant pyridine nucleotide-dependent hydrogenases. This complex, which is homologous to the SH from R. eutropha, has revealed advantages for spectroscopic analysis in earlier studies. Here it was characterized biochemically and spectroscopically for the first time in direct comparison with the SH from R. eutropha.