As flavoproteinas AhpF (Alquil Hidroperoxido Redutase subunidade F) e TrxR (Tiorredoxina Redutase) sao membros da familia piridina dissulfeto redutase e possuem atividade dissulfeto redutase as custas de NAD(P)H. A proteina TrxR e responsavel pela reducao de Tiorredoxina (Trx) que participa do ciclo catalitico de grande parte das enzimas da familia peroxirredoxina, alem de outras enzimas. AhpF e dedicada a reducao de AhpC, (Alquil Hidroperoxido Redutase subunidade C) uma peroxirredoxina exclusiva de bacterias, AhpC e AhpF juntas formam sistema AhpR (Alquil Hidroperoxido Redutase). AhpF possui dois dominios, Trx-like (N-terminal) e TrxR-like (C-terminal); sendo que a ultima possui alta similaridade de estrutura e sequencia a TrxR. De forma interessante AhpF possui atividade NADH-oxidase formadora de H2O2, enquanto TrxR nao possui. Provavelmente pequenas mudancas na sua estrutura contribuem para essa diferenca. A atividade NADH-oxidase esta presente em algumas flavoproteinas e esta geralmente centrada no anel de isoaloxazina do cofator FAD. Este anel quando no estado radicalar e chamado de semiquinona. A semiquinona pode estar protonada ou desprotonada, sendo que a ultima forma e relacionada com atividades oxidase e oxigenase de maneira geral, mas ainda nao existem regras claras a respeito da reatividade de flavoproteinas com o oxigenio. Para elucidar quais poderiam ser essas diferencas, nos clonamos os genes para as proteinas TrxR, AhpC e AhpF da bacteria fitopatogenica Xylella fastidiosa e os expressamos em Escherichia coli. Reconstituimos com sucesso o sistema AhpR in vitro, medindo o consumo de H2O2 na presenca de AhpC, AhpF e NADH atraves de eletrodos especificos para peroxido. Da mesma forma caracterizamos a atividade NADH-oxidase de AhpF medindo o consumo de oxigenio e a producao de peroxido de hidrogenio. Alem disso, demonstramos que a atividade NAD(P)H-oxidase e ausente em TrxR atraves de ensaios de consumo de NADH. A expressao de somente o dominio C-terminal de AhpF manteve a atividade NADH-oxidase como na proteina selvagem, levando a crer que sao diferencas no motivo TrxR-like que disparam a atividade NADH-oxidase. Analisando a sobreposicao de estruturas tridimensionais de AhpF e TrxR disponiveis no PDB em conjunto com o alinhamento de sequencia de aminoacidos destas proteinas em diferentes organismos, identificamos tres possiveis candidatos que poderiam estar envolvidos na atividade NADH-oxidase. Atraves de mutacao sitio dirigida, identificamos que a retirada do residuo de histidina entre o motivo CXXC de AhpF fez o mutante AhpF H347T apresentar metade da atividade especifica NADHoxidase. Da mesma forma a mutacao reversa em TrxR, adicionando o residuo de histidina no motivo CXXC levou a TrxR T142H apresentar uma atividade especifica significativamente maior que a TrxR selvagem. Os dados em conjunto sugerem que o residuo de histidina por sua natureza polar e relevante para a desprotonacao do anel de izoaloxazina do FAD, e a sua consequente reatividade com o oxigenio, sendo um fator importante para a atividade NADH-oxidase presente em AhpF / The flavoproteins AhpF (Alkylhydroperoxide reductase subunit F) and TrxR (Thioredoxin Reductase) are members of the nucleotide pyridine disulfide oxidoreductase family and possess disulfide reductase activity at the expense of NAD(P)H. The TrxR protein is responsible for the reduction of thioredoxin (Trx), which is used in the catalytic cycle of most peroxiredoxins, among other enzymes. AhpF is dedicated to reducing the bacterial peroxiredoxin AhpC, and together they form the AhpR system (Alkylhydroperoxide reductase system). AhpF has two domains, Trx-like (N-terminal) and TrxR-like (C-terminal); the latter has high of similarity to TrxR. Intriguingly, AhpF has an H2O2-forming NAD(P)H-dependent oxidase activity, while TrxR does not. Slight changes in the structures of these two enzymes probably account for this phenomenon. The NADH-oxidase activity is present in some flavoproteins and is generally centered in the isoalloxazine ring of the FAD cofactor. This ring in its radical state is called a semiquinone, which can be protonated or deprotonated. The deprotonated form is related to oxidase and oxygenase activities, but there are no clear rules as to the reactivity of flavoproteins and molecular oxygen. In order to elucidate these differences, we have cloned genes which code the proteins TrxR, AhpC and AhpF of the phytopathogenic bacteria Xylella fastidiosa and have expressed them in Escherichia coli. We have successfully reconstituted the AhpR system in vitro, measuring the H2O2 decrease in the presence of AhpC, AhpF and NADH, by using specific electrodes. We have similarly characterized the NADH-oxidase activity of AhpF by measuring the decrease in the levels of oxygen and the production of hydrogen peroxide. Furthermore, through assays measuring the consumption of NADH, we have demonstrated that the NAD(P)H-oxidase activity is non-existent in TrxR. The expression of only the C-terminal domain of AhpF showed NADH-oxidase activity similar to the wild-type protein, which indicated that the NADH-oxidase activity occurs due to differences in the TrxR-like motif. By analyzing the superposition of the threedimensional structures of AhpF and TrxR, as well as the alignment of their amino acid sequence in different organisms, we identified three possible candidates which could be involved in NADH-oxidase activity. Through site-directed mutation, we found that the removal of the histidine residue within the CXXC motif of AhpF caused the mutant AhpF H347T to present half of the NADH-oxidase specific activity, when compared to the wild-type protein. Likewise, the reverse mutation in TrxR, adding the histidine residue to the CXXC motif, caused TrxR T142H to have a significantly higher specific activity when compared to the wild-type TrxR. The data suggests that, because of its polar nature, the histidine residue is relevant to the deprotonation of the isoalloxazine ring of FAD and its reactivity to oxygen, and, therefore, is an important factor to the NADHoxidase activity of AhpF
| Identifer | oai:union.ndltd.org:IBICT/oai:teses.usp.br:tde-11012013-160639 |
| Date | 27 September 2012 |
| Creators | Marcela Valente Pimenta |
| Contributors | Luis Eduardo Soares Netto, Marilis do Valle Marques, Marcos Antonio de Oliveira |
| Publisher | Universidade de São Paulo, Ciências Biológicas (Biologia Genética), USP, BR |
| Source Sets | IBICT Brazilian ETDs |
| Language | Portuguese |
| Detected Language | English |
| Type | info:eu-repo/semantics/publishedVersion, info:eu-repo/semantics/masterThesis |
| Source | reponame:Biblioteca Digital de Teses e Dissertações da USP, instname:Universidade de São Paulo, instacron:USP |
| Rights | info:eu-repo/semantics/openAccess |
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