57
activates a specific amino acid such as its acyl adenylate by reaction with ATP. This
activated ester is then covalently connected as its thioester in the thiolation domain
(T). The condensation domain (C) catalyzes the direct transfer to another intermediate
of acyl amino acid in the adjacent posterior form to form a peptide bond. The NRPS
synthesizes both the chromophores (Mossialos et al. 2002) and the peptide chains of
pyoverdine (Crosa and Walsh 2002). The generated NRPSs are very different: they are
mostly part of proteogenic and non-proteogenic amino acids and can be linear or
branched cyclically with a variable length. They show extensive secondary modification after its synthesis outside from the ribosome. Many fungal NRPSs have a high
economic and/or ecological value such as β-lactam antibiotics, the immunosuppressant Cyclosporin A, but also mycotoxins such as glyoxin.
4.3.2.1 NRPS-Dependent Biosynthesis
ATP pyrophosphate is the main enzyme involved in this process, which is widely used
in exchange assays and to determine the substrate specificity of the adenylation
domains within the multienzyme synthetase. The formation of hydroxy acid by capturing the activated carboxyl group with hydroxylamine is an alternative method of
analysis of the enzymes involved in the formation of acyl adenylate. Hydroxamic acid
can be converted into its ferric complex and can be detected spectrophotometrically.
The NRPS is a set of peptides of broad structural diversity and biological activity. For the recognition, activation, and modification of each incorporated amino,
they have a different unitary structure and each unit is responsible for its specific
function to finally form a peptide product (Lautru 2004). The number and order of
units in the PRPS are responsible for determing their size and sequence.
NRPS is an important mechanism for the biosynthesis of extra- and intracellular
iron chelating siderophores that help the growth of fungi (Eisendle et al. 2006). In
A. fumigatus, the studies identified the expression of three NRP synthetase genes
(sidC, D, and E), which are, at various levels, susceptible to regulation by the level
of free iron present in the culture medium. It has been shown that the expression of
sidD is significantly upregulated in culture medium that limit iron (iron free or
20 mM free iron), concomitant with the production of siderophores and that the
related sidD protein is present, as determined using the combined approach of
2D-PAGE/MALDI-TOF and TOF/TOF mass spectrometry. Previous studies on the
expression of microarray in S. cerevisiae have shown that the disabled Open Reading
Frame (ORF) can be expressed at the level of transcription (Harrison et al. 2002).
Therefore, functional identification of NRP synthetases, at the protein level, has
been important since then pseudogenes may undergo transcription due to the presence of functional promoters (Lee et al. 2005). Furthermore, the identification of
NRP synthase gene expression can be problematic (Cramer et al. 2006). Significantly,
one gene (NPS6) has been identified in the pathogenic fungus of the Cochliobolus
heterostrophus plant that contributes both to the virulence of the fungi in maize and
resistance to oxidative stress (Lee et al. 2005). The elimination of NPS6 in the plant
pathogenic ascomycetes Alternaria brassicicola, Cochliobolus miyabeanus, C. heterostrophus, and F. graminearum produced a reduced virulence and a greater sensitivity to H 2 O 2 (Oide et al. 2006).
4 Siderophores: Mediated Iron Acquisition and Virulence of Brown Rot Disease…
activates a specific amino acid such as its acyl adenylate by reaction with ATP. This
activated ester is then covalently connected as its thioester in the thiolation domain
(T). The condensation domain (C) catalyzes the direct transfer to another intermediate
of acyl amino acid in the adjacent posterior form to form a peptide bond. The NRPS
synthesizes both the chromophores (Mossialos et al. 2002) and the peptide chains of
pyoverdine (Crosa and Walsh 2002). The generated NRPSs are very different: they are
mostly part of proteogenic and non-proteogenic amino acids and can be linear or
branched cyclically with a variable length. They show extensive secondary modification after its synthesis outside from the ribosome. Many fungal NRPSs have a high
economic and/or ecological value such as β-lactam antibiotics, the immunosuppressant Cyclosporin A, but also mycotoxins such as glyoxin.
4.3.2.1 NRPS-Dependent Biosynthesis
ATP pyrophosphate is the main enzyme involved in this process, which is widely used
in exchange assays and to determine the substrate specificity of the adenylation
domains within the multienzyme synthetase. The formation of hydroxy acid by capturing the activated carboxyl group with hydroxylamine is an alternative method of
analysis of the enzymes involved in the formation of acyl adenylate. Hydroxamic acid
can be converted into its ferric complex and can be detected spectrophotometrically.
The NRPS is a set of peptides of broad structural diversity and biological activity. For the recognition, activation, and modification of each incorporated amino,
they have a different unitary structure and each unit is responsible for its specific
function to finally form a peptide product (Lautru 2004). The number and order of
units in the PRPS are responsible for determing their size and sequence.
NRPS is an important mechanism for the biosynthesis of extra- and intracellular
iron chelating siderophores that help the growth of fungi (Eisendle et al. 2006). In
A. fumigatus, the studies identified the expression of three NRP synthetase genes
(sidC, D, and E), which are, at various levels, susceptible to regulation by the level
of free iron present in the culture medium. It has been shown that the expression of
sidD is significantly upregulated in culture medium that limit iron (iron free or
20 mM free iron), concomitant with the production of siderophores and that the
related sidD protein is present, as determined using the combined approach of
2D-PAGE/MALDI-TOF and TOF/TOF mass spectrometry. Previous studies on the
expression of microarray in S. cerevisiae have shown that the disabled Open Reading
Frame (ORF) can be expressed at the level of transcription (Harrison et al. 2002).
Therefore, functional identification of NRP synthetases, at the protein level, has
been important since then pseudogenes may undergo transcription due to the presence of functional promoters (Lee et al. 2005). Furthermore, the identification of
NRP synthase gene expression can be problematic (Cramer et al. 2006). Significantly,
one gene (NPS6) has been identified in the pathogenic fungus of the Cochliobolus
heterostrophus plant that contributes both to the virulence of the fungi in maize and
resistance to oxidative stress (Lee et al. 2005). The elimination of NPS6 in the plant
pathogenic ascomycetes Alternaria brassicicola, Cochliobolus miyabeanus, C. heterostrophus, and F. graminearum produced a reduced virulence and a greater sensitivity to H 2 O 2 (Oide et al. 2006).
4 Siderophores: Mediated Iron Acquisition and Virulence of Brown Rot Disease…