STRESS RESPONSES - Cyanobacteria - page 701

PCC 7421, S . elongatus PCC 6301, S . elongatus PCC 7942, C . watsonii WH8501, Cyanothece sp. PCC 8801
and N . punctiforme PCC 73102) except in case of Synechocystis sp. stain PCC 6803 and two strains of
Synechococcus which have two paralogues. In the rest of the cyanobacterial genomes investigated
[ M. aeruginosa NIES-843, two strains of Synechococcus , T . elongatus BP-1, A . marina MBIC11017, strains
of Cyaonothece (ATCC 51142, CCY0110 and PCC 7424), Lyngbya sp. PCC 8106, T . erythraeum IMS101,
A . variabilis ATCC 29413, No . spunimgena CCY9414, Anabaena sp. strain PCC 7120] the GPx gene
sequence is absent. The cynobacterial GPx have a conserved active site with three cysteine residues
from N- to C-terminus, XCG, -PCN- and FCY, respectively with the latter being absent in Synechocystis
sp. strain PCC 6803 and Synechococcus PCC 7002 GPx proteins (Bernroitner et al ., 2009).
G) Glutaredoxins (Grxs): These are small glutathione-disulphide oxodoreductases belonging to
the Trx-fold superfamily (Martin, 1995). These are present in bacteria, plants and mammals. The
importance of Grx was recognized by Holmgren (1976) who fi rst reported its requirement as an
alternative hydrogen donor in the reduction of intramolecular disulphide in ribonucleotide reductase,
an important enzyme for DNA synthesis. Three major groups of Grxs are known. The classical type
Grx is a small 10 kDa protein with an active site of Cys-Pro-Tyr-Cys. E . coli (Grx1 and Grx3) and
yeast (Grx1 and Grx2) type Grxs belong to this group. The second group possesses an active site of
Cys-Gly-Phe-Ser and yeast Grxs (Grx3, Grx4 and Grx5) belong to this group (Rodriguez-Manzaneque
et al ., 1999). E . coli Grx2 that is structurally related to glutathione-S-transferase belongs to the third
group (Xia et al ., 2001). The Grxs catalyze reduction of protein disulphides through dithiol mechanism
and GSH-protein mixed disulphides through monothiol mechanism in a coupled system with GSH,
NADPH and GR and maintain the redox homoeostasis (Holmgren, 1989; Holmgren and Aslund,
1995). In dithiol mechanism both cysteines of the conserved motif are required for the dithiol redox
reaction whereas in the monothiol mechanism only one cysteine of the active site is involved.
Besides DNA synthesis, a number of cellular functions are infl uenced by Grxs such as regulation
of transcription factors, reduction of dehydroascorbate, protein folding and sulphur metabolism
(Wells et al ., 1993; Fernandes and Holmgren, 2004). The functions of Grxs stretch far beyond a simple
Trx backup system as these enzymes are effi cient in the reactivation of proteins inactivated during
oxidative stress. Evidences in favour of the protection conferred by Grxs against oxidative stress have
been put forward in the reactivation of HIV-1 protease (Davis et al ., 1997) and eukaryotic nuclear
factor I (Bandyopadhyay et al ., 1998).
The protective role of Grxs against oxidative stress in cyanobacteria has been investigated. The
completely sequenced genome of Synechocystis sp. strain PCC 6803 revealed the presence three
probable genes of Grxs (Kaneko et al ., 1996). One of these, ORF ssr2061 (of 264 codons) has been
cloned and the deduced amino acid sequence of the Grx shares 29-42% identity in amino acid
sequence of Grxs from E . coli (35%), yeast (31%), rice (42%) and human (29%). E . coli BL21 (DE3)
cells transformed with pET-2061 vector overexpressed the recombinant protein (Grx2061; up to 20%
of the total protein quantifi ed by band scan) and the purifi ed protein exhibited NADPH oxidation
in the 2-hydroxyethyldisulphide assay. However, in the active site of Grx2061 between the active
Cys residues, Tyr residue was replaced by Phe residue as observed in case of rice but in the rest the
conserved active site is present. Higher Grx activity in the transformant enabled it to grow in presence
of H 2 O 2 . The protective role of overexpressed Grx in E . coli cells against oxidative stress induced
by H 2 O 2 has been highlighted (Li et al ., 2005). Deletion of ssr2061 was not lethal to Synechocystis sp.
strain PCC 6803 but reduced its viability which indicates that ssr2061 has a protective role against
oxidative stress. A mixed disulphide affi nity approach was undertaken to identify the target proteins
interacting with Grx2061 in vivo . By employing mutated Grx2061 protein (at the second Cys residue