Full text
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| Author(s): |
Marcela Valente Pimenta
Total Authors: 1
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| Document type: | Master's Dissertation |
| Press: | São Paulo. |
| Institution: | Universidade de São Paulo (USP). Instituto de Biociências (IBIOC/SB) |
| Defense date: | 2012-09-27 |
| Examining board members: |
Luis Eduardo Soares Netto;
Marilis do Valle Marques;
Marcos Antonio de Oliveira
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| Advisor: | Luis Eduardo Soares Netto |
| Abstract | |
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 (AU) | |
| FAPESP's process: | 09/06833-9 - Caracterization of the biological activity of the AhpR system of Xylella fastidiosa |
| Grantee: | Marcela Valente Pimenta |
| Support Opportunities: | Scholarships in Brazil - Master |