form, a homodimer formed by subunits of approximately 35 kDa with NADPH,
FAD, and central domains, the enzyme from mammals has a larger molecular mass
(approximately 55 kDa) and contains selenocysteine, which is active in catalysis
(Arscott et al. 1997). In plants, the first NTR cloned and characterized, from
Arabidopsis (Jacquot et al. 1994) and wheat (Serrato et al. 2002), revealed that the
enzyme is similar to the one in prokaryotes.
When the genome sequences from Arabidopsis and rice were available, a search
of the genes encoding NTR in these plants revealed the presence of three genes
termed NTRA, NTRB, and NTRC (Serrato et al. 2002). The NTRA and NTRB genes
encode polypeptides corresponding to those previously reported from Arabidopsis
and wheat. Further analyses in Arabidopsis revealed that NTRB is the predominant
isoform in mitochondria whereas NTRA is predominant in the cytosol (Reichheld
et al. 2005). The polypeptide deduced of the NTRC gene contained the expected
active site double Cys as well as the NADPH and FAD domains, hence being very
similar to NTRA and NTRB. However, in contrast to these proteins, the deduced
NTRC polypeptide contained a putative transit peptide at the N-terminus and, most
intriguingly, a putative TRX domain at the C-terminus; thus, it contained a complete
NTR-TRX system in a single polypeptide (Serrato et al. 2004). Both the NTR and
TRX domains were expressed in the bacterium Escherichia coli as N-terminal
His-tagged proteins, and the biochemical analysis confirmed that these truncated
polypeptides displayed NTR and TRX activity, respectively, in vitro, leading to the
proposal that NTRC is a bifunctional enzyme (Serrato et al. 2004). Moreover, the
search in genomes available at the moment, and recently confirmed, showed that
the NTRC gene is exclusively found in aerobic photosynthetic organisms including
some, but not all, cyanobacteria, algae, and plants (Serrato et al. 2004; Najera et al.
2017). Furthermore, it was shown that NTRC is localized in the chloroplast stroma
both in Arabidopsis and rice (Serrato et al. 2004; Moon et al. 2006), hence implying
that the putative transit peptide deduced of the NTRC gene sequence actually serves
to target the enzyme to this organelle. More recently, the localization of NTRC was
analyzed by expressing NTRC::GFP fusion proteins in Arabidopsis. This study
confirmed the localization of NTRC in chloroplasts and showed the presence of
the enzyme in plastids from non-photosynthetic tissues such as root amyloplasts,
though NTRC is much less abundant in these tissues as compared with green tissues
(Kirchsteiger et al. 2012). The phylogenetic analysis suggests that the NTRC gene
originated in cyanobacteria most probably by mutational joining of adjacent but
independent NTR and TRX genes. The combination of NTR and TRX activities in a
single enzyme might provide selective advantage, which explains the presence of the
NTRC gene in all eukaryotic photosynthetic organisms (Najera et al. 2017).
2.2.2 NTRC Is an Efficient Reductant of 2-Cys PRX
An important breakthrough for the biochemical characterization of NTRC was the
identification of 2-Cys PRX as a target of the enzyme. 2-Cys PRX are thioldependent PRX able to reduce hydrogen or organic peroxides to water or the
232
I. Thormählen et al.
FAD, and central domains, the enzyme from mammals has a larger molecular mass
(approximately 55 kDa) and contains selenocysteine, which is active in catalysis
(Arscott et al. 1997). In plants, the first NTR cloned and characterized, from
Arabidopsis (Jacquot et al. 1994) and wheat (Serrato et al. 2002), revealed that the
enzyme is similar to the one in prokaryotes.
When the genome sequences from Arabidopsis and rice were available, a search
of the genes encoding NTR in these plants revealed the presence of three genes
termed NTRA, NTRB, and NTRC (Serrato et al. 2002). The NTRA and NTRB genes
encode polypeptides corresponding to those previously reported from Arabidopsis
and wheat. Further analyses in Arabidopsis revealed that NTRB is the predominant
isoform in mitochondria whereas NTRA is predominant in the cytosol (Reichheld
et al. 2005). The polypeptide deduced of the NTRC gene contained the expected
active site double Cys as well as the NADPH and FAD domains, hence being very
similar to NTRA and NTRB. However, in contrast to these proteins, the deduced
NTRC polypeptide contained a putative transit peptide at the N-terminus and, most
intriguingly, a putative TRX domain at the C-terminus; thus, it contained a complete
NTR-TRX system in a single polypeptide (Serrato et al. 2004). Both the NTR and
TRX domains were expressed in the bacterium Escherichia coli as N-terminal
His-tagged proteins, and the biochemical analysis confirmed that these truncated
polypeptides displayed NTR and TRX activity, respectively, in vitro, leading to the
proposal that NTRC is a bifunctional enzyme (Serrato et al. 2004). Moreover, the
search in genomes available at the moment, and recently confirmed, showed that
the NTRC gene is exclusively found in aerobic photosynthetic organisms including
some, but not all, cyanobacteria, algae, and plants (Serrato et al. 2004; Najera et al.
2017). Furthermore, it was shown that NTRC is localized in the chloroplast stroma
both in Arabidopsis and rice (Serrato et al. 2004; Moon et al. 2006), hence implying
that the putative transit peptide deduced of the NTRC gene sequence actually serves
to target the enzyme to this organelle. More recently, the localization of NTRC was
analyzed by expressing NTRC::GFP fusion proteins in Arabidopsis. This study
confirmed the localization of NTRC in chloroplasts and showed the presence of
the enzyme in plastids from non-photosynthetic tissues such as root amyloplasts,
though NTRC is much less abundant in these tissues as compared with green tissues
(Kirchsteiger et al. 2012). The phylogenetic analysis suggests that the NTRC gene
originated in cyanobacteria most probably by mutational joining of adjacent but
independent NTR and TRX genes. The combination of NTR and TRX activities in a
single enzyme might provide selective advantage, which explains the presence of the
NTRC gene in all eukaryotic photosynthetic organisms (Najera et al. 2017).
2.2.2 NTRC Is an Efficient Reductant of 2-Cys PRX
An important breakthrough for the biochemical characterization of NTRC was the
identification of 2-Cys PRX as a target of the enzyme. 2-Cys PRX are thioldependent PRX able to reduce hydrogen or organic peroxides to water or the
232
I. Thormählen et al.