| Authors: |
Matilde Maiorino
and Fulvio Ursini
and Valentina Bosello
and Stefano Toppo
and Silvio C. E. Tosatto
and Pierlulgi Mauri
and Katja Becker
and Antonella Roveri
and Cristiana Bulato
and Louise Benazzi
and Antonella de Palma
and Leopold Flohe
|
| Tags: |
IFZ
glutathione_peroxidase
mechanism_of_action
peroxiredoxin
selenocysteine
thioredoxin_peroxidase
|
| Abstract: |
Some members of the glutathione peroxidase (GPx) family have been
reported to accept thioredoxin as reducing substrate. However, the
selenocysteine-containing ones oxidise thioredoxin (Trx), if at all, at
extremely slow rates. In contrast, the Cys homolog of Drosophila
melanogaster exhibits a clear preference for Trx, the net forward rate
constant, k�(+2), for reduction by Trx being 1.5 x 10(6) M-1 s(-1), but
only 5.4 M-1 s(-1) for glutathione. Like other CysGPxs with thioredoxin
peroxidase activity, Drosophila melanogaster (Dm)GPx oxidized by H2O2
contained an intra-molecular disulfide bridge between the active-site
cysteine (C45; C-P) and C91. Site-directed mutagenesis of C91 in DmGPx
abrogated Trx peroxidase activity, but increased the rate constant for
glutathione by two orders of magnitude. In contrast, a replacement of
C74 by Ser or Ala only marginally affected activity and specificity of
DmGPx. Furthermore, LCMS/MS analysis of oxidized DmGPx exposed to a
reduced Trx C35S mutant yielded a dead-end intermediate containing a
disulfide between Trx C32 and DmGPx C91. Thus, the catalytic mechanism
of DmGPx, unlike that of selenocysteine (Sec)GPxs, involves formation
of an internal disulfide that is pivotal to the interaction with Trx.
Hereby C91, like the analogous second cysteine in 2-cysteine
peroxiredoxins, adopts the role of a ��resolving" cysteine (C-R).
Molecular modeling and homology considerations based on 450 GPxs
suggest peculiar features to determine Trx specificity: (i) a
nonaligned second Cys within the fourth helix that acts as C-R; (ii)
deletions of the subunit interfaces typical of tetrameric GPxs leading
to flexibility of the C-R-Containing loop. Based of these
characteristics, most of the nonmammalian CysGPxs, in functional terms,
are thioredoxin peroxidases. (c) 2006 Elsevier Ltd. All rights reserved. |
@article{ISI:000243561600012,
title = {The thioredoxin specificity of Drosophila GPx: A paradigm for a peroxiredoxin-like mechanism of many glutathione peroxidases},
author = {Matilde Maiorino and Fulvio Ursini and Valentina Bosello and Stefano Toppo and Silvio C. E. Tosatto and Pierlulgi Mauri and Katja Becker and Antonella Roveri and Cristiana Bulato and Louise Benazzi and Antonella de Palma and Leopold Flohe},
journal = {JOURNAL OF MOLECULAR BIOLOGY},
number = {4},
pages = {1033-1046},
volume = {365},
year = {2007},
abstract = {Some members of the glutathione peroxidase (GPx) family have been
reported to accept thioredoxin as reducing substrate. However, the
selenocysteine-containing ones oxidise thioredoxin (Trx), if at all, at
extremely slow rates. In contrast, the Cys homolog of Drosophila
melanogaster exhibits a clear preference for Trx, the net forward rate
constant, k�(+2), for reduction by Trx being 1.5 x 10(6) M-1 s(-1), but
only 5.4 M-1 s(-1) for glutathione. Like other CysGPxs with thioredoxin
peroxidase activity, Drosophila melanogaster (Dm)GPx oxidized by H2O2
contained an intra-molecular disulfide bridge between the active-site
cysteine (C45; C-P) and C91. Site-directed mutagenesis of C91 in DmGPx
abrogated Trx peroxidase activity, but increased the rate constant for
glutathione by two orders of magnitude. In contrast, a replacement of
C74 by Ser or Ala only marginally affected activity and specificity of
DmGPx. Furthermore, LCMS/MS analysis of oxidized DmGPx exposed to a
reduced Trx C35S mutant yielded a dead-end intermediate containing a
disulfide between Trx C32 and DmGPx C91. Thus, the catalytic mechanism
of DmGPx, unlike that of selenocysteine (Sec)GPxs, involves formation
of an internal disulfide that is pivotal to the interaction with Trx.
Hereby C91, like the analogous second cysteine in 2-cysteine
peroxiredoxins, adopts the role of a ��resolving" cysteine (C-R).
Molecular modeling and homology considerations based on 450 GPxs
suggest peculiar features to determine Trx specificity: (i) a
nonaligned second Cys within the fourth helix that acts as C-R; (ii)
deletions of the subunit interfaces typical of tetrameric GPxs leading
to flexibility of the C-R-Containing loop. Based of these
characteristics, most of the nonmammalian CysGPxs, in functional terms,
are thioredoxin peroxidases. (c) 2006 Elsevier Ltd. All rights reserved.},
issn = {0022-2836},
keywords = {IFZ glutathione_peroxidase mechanism_of_action peroxiredoxin selenocysteine thioredoxin_peroxidase }
}
::