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1. Introduction:
1.1 Cyanobacteria:
The cyanobacteria are an ecologically, morphologically, and physiologically
Gram negative bacteria, but their mode of nutrition is photoautorphic. Like higher
plants they possess chlorophyll a and water soluble red and blue
phycobiliproteins as well as phtosystem I and II, hence they can use water for
atmosphere. Along with this beneficial part of cyanobacteria they were also
were documented since the late 19th century[4]. These reports describes
sickness and death of livestock, pets and wildlife following ingestion of water
containing toxic algae cells or the toxins released by ageing cells. Most recent
products began with the pioneering work of Richard Moore at the University of
Hawaii. In the early 1970ʼs his laboratory published several surveys of marine
cyanobacteria from the Pacific showing that they were rich in potential anticancer
The genomic revolution has changed the face of natural product research. Over
the last two decades, more than 150 complete biosynthetic gene clusters from
over the past 15 years into the genetics studies of secondary metabolites
towards using the molecular genetics to identify biosynthetic pathways and novel
enzymes. The principal pioneer in this area was Sir David Hopwood who
were sequenced (a formidable task during those days) and the primary sequence
metabolites, only few biosynthetic studies have been completed (table __)[81,82].
Consequently, very little has been known about the molecular mechanism and
representative species, hence it has been stressed that cyanobacteria are the
most unlucky organisms having great potential and economic values but poorly
characterized[87].
bioactive peptides in all growth phases[88], but depending upon the growth
Molecules made by NRPS are generally cyclic, have high density of high
proteinogenic amino acids and these amino acids are often connected by bonds
other than peptide and disulphide bonds. NRPS are known to be very large
proteins and consists of series of repeating enzymes fused together. Such fusion
acid building block is incorporated into the peptide product by each module,
NRPS with ten modules stitched together. This is called as Structural Colinearity.
Each module is normally specific to a particular amino acid substrate but this rule
organized into modules, each of the modules are responsible for one cycle of
elongation by the incorporation of single amino acid into the chain. Each
amino acid and activates it as an amino acyl adenylate. The activated amino
protein (PCP) or thiolation domain (T). Condensation domain (C) catalyze the
peptide bond formation between amino acid in adjacent module. The chain is
domain (TE). Apart from these basic modules, which are ubiquitously present,
_____________________________________________________Introduction______
there also present certain tailoring/ accessory modules which certainly adds to
methylation and D-amino acids generally not found in any other system in nature
[94].
modules, one for each amino acid to be built into the peptide product. Generally
the modules are colinear to the sequence of the synthesized peptide, thus
A typical module comprises 1000 residues and is responsible for one reaction
domain, responsible for substrate selection and activation through ATP hydrolysis
[97,98], a 10 kDa downstream peptidyl carrier protein (PCP) domain for the
inserted at specific locations in the module [89]. This enlarges the broad
(FAS) and polyketide synthesis (PKS), which are both carried out on similarly
the PCP domain, the acyl carrier proteins (ACPs) of PKS and FAS. The cofactor
Adenylation Domain:
Adenylation domain catalyzes the specific activation of carboxyl group of amino
acid, imino acids or hydroxyl acids. Each adenylation domain has a specific
geometry of binding pocket which only allows a specific amino acid to enter into
the catalytic site. The analysis of phenylalanine binding pocket of the first module
of the Bacillus brevis Gramicidin S synthetase I (GrsA) has led to the prediction of
expressed as a single domain and codes for the initiation module at the putative
domain border between the A and PCP domain. The A domain has same
homology in its chemistry that of ribosomal pathway aminoacyl tRNA but has no
is non-covalently attached to the A-domain (red). The thiol group of the 4’PP
cofactor of the PCP domain (green) accepts the activated substrate. In the next
step is the formation of first peptide bond which is catalyzed by the C domain
loaded cofactor of the PCP domain to a nucleophile acceptor position “a” and
module (aa1) to an electrophile donor position “d” of this C domain are necessary
for the reaction to take place. The result of this reaction is the formation of an
elongated peptide loaded on to the PCP domain and recycling of the upstream
PCP thiol group. The peptide linked to the PCP domain is then translocated to
the third position to be served, the electrophile donor position of the downstream
C domain. The second pepbond bond is formed here (reaction 4) with the amino
acid activated by the following A domain (aa3) which is fixed to the corresponding
downstream PCP. After completion of this reaction cycle, the growing peptide
adopts a regenerated status (thiol). The 4'PP cofactor of the PCP domain is
shown in the three positions that have to be served; there is only one cofactor for
The main difference between the ribosomal and nonribosomal systems is the
mechanism and because of the presence of A domain for each residue added
into the growing peptide chain a relative relaxed substrate selectivity has been
domain of NosA activates Val, Ile and Leu when expressed in E. coli, but Leu is
domains have been identified so far. They are generally present in NRPS
This is the second domain generally found immediately after the A domain. The
domain for aminoacyl and peptidyl elongation cycle. These domains also work in
(T) is also called as Peptidyl Carrier Protein (PCP). Its function is more or less
similar to that of ACP (acyl carrier protein) of the PKS system. Although ACP
and PCP are functionally similar, they show little homology except at the cofactor
activates their substrate as acyl adenylate and fix them for further treatment as a
_____________________________________________________Introduction______
thioester to the 4 ‘PP cofactor of the carrier protein[110,111]. Besides the PCP
domain structure, the NMR structures of prototypes for FAS ACPs and PKS
ACPs are known. All three carrier proteins (FAS ACP, PKS ACP, and PCP)
antiparallel four-helix bundle with a long loop between the first two helices (fig:
___). The serine residue which is the site of cofactor binding is located at the
Cartoon structure of (a) the NRPS PCP domain (PDB code 1DNY), (b) the fatty acid synthase ACP (PDB code 1ACP), and (c)
The
the primary
actinorhodin role
polyketide synthaseof
ACP PCP
(PDB code domain
1AF8). Theis inserine
invariant theresidues
transport ofcofactor are
that carry the 4′PP
intermediates which are activated by the adenylation domain
highlighted in ball-and-stick format. The similarity of the overall fold as well as differences in lengths and relative orientations of
the helices between these members of the same protein family are apparent. (The figure was taken from Weber& Marahiel[2])
and subsequent interaction with the condensation domain for
aminoacyl and peptidyl elongation cycle[112].
This domain is the third domain present in the NRPS system. It catalyze the
arm of the T/PCP domain (which is present upstream) to the amino acid bound to
the downstream T domain[113]. This is the reason the first module usually do not
contains C domain but the second module has the domain sequence CAT
domains are inserted between each consecutive pair of activating units (which
may include additional auxillary domains such as E, N-Met) (Fig: ___). This
_____________________________________________________Introduction______
arrangement resembles the basic setup for the sequential linkage of activating
amino acids to synthesize a linear peptide. Thus it can be said that the number
number of the linear intermediates[96]. Not much information has been available
for the C domain up until now. According to Raush[114], there exists 7 functional
formation between two L-amino acids. ii) DCL domain which links an L-amino acid
to a growing peptide chain ending with a D-amino acid. iii) C domain starter unit
generally acylates the first amino acid with a β-hydroxy-carboxylic acid (typically
the last amino acid in the growing peptide. According to Raush[114], there also
exists a Dual E/C domains which catalyze both epimerization and condensation
reactions.
Figure___: Module and domain structure of NRPS: Complete NRPS consisting of three modules viz,
initiation, elongation and termination. Condensation domain (C) showing approximate positions of the seven
motifs. Other principal and ancillary domains such as Adenylation domain (A domain), N-Meth: N-methylation
domain (optional – does not appear in all NRPS), PCP: Thiolation domain (T domain or Peptidyl Carrier
Protein domain), Epi: Epimerization domain (optional). Other optional domains are: Heterocyclization,
Oxidation, Reduction and Formylation domain (modified from Rausch[114])
_____________________________________________________Introduction______
The TE domain is about 250 amino acid residue located to the C-terminal end
which is primarily involved in the addition of the last amino acid to the linear
growing peptide chain. This domain has been found in the same location in the
its strategic location, it can be said that the TE domain might involved in
peptide biosynthesis. The TE domain generally has a core motif of GxSxG which
residue in length and show great homology to the TE domain involved in the fatty
acid biosynthesis of the mammalian cells. Thus it can be inferred that TE domain
A) Epimerization domains:
epimerize aminoacyl and peptidyl intermediates at the thioester stage and this
reaction is reversible thus they can maintain a state of equilibrium between these
two isomers.
C) N-methylation domain:
formation of the primed amino acid. This was first found in the fungal system
integrated with the A domain between the core motif A8-A9. this domain is about
450 amino acid long and it shares sequence similarities to the S-adenosyl-L-
insertion or deletion[117].
_____________________________________________________Introduction______
D) Oxidation domain:
These domains are generally 200 amino acid residue showing sequence
homolog to the DNA binding proteins. They are generally present in adenylation
domains between the core motif A8-A9. these domains are found in
speculated that BarI and BarJ has been involved in the oxidative
decarboxylation[17].
E) Reduction domain:
The reduction domain is about 400 amino acid long showing significant similarity
incorporation of more than one amino acid which is greatly responsible for the
of the residues in these positions for the function of the product. The A domain
substrate[105,107]. A deep insight into the substrate binding was revealed when
acids for adenylation domains. He also provided general rules for inferring the
specificity codes from the amino acid sequences of adenylation domains. Chang
et al.[17] showed that the activity assay of adenylation domains of barD, barE
specificity.
Generally, in NRPS gene clusters the order of the coded activities is colinear with
the structure of the product, and the number of modules is the same as the
the peptide, provided the substrate specificities of the adenylation domains are
can be deduced from the gene sequence. This is made possible by comparing
_____________________________________________________Introduction______
from a strain that produces more than one nonribosomal peptide. Currently,
peptides are assembled by the iterative use of modules or domains, so that the
peptide chain is composed of smaller repeated units. Examples of this type are
from Brevibacillus brevis [129]. The activities and number of modules correspond
Polyketides (PKS) are large multifunctional protein complexes which catalyze the
modular organization and each module carries all essential information for the
thioester derivative of carboxylic acid into the growing chain. The number of
_____________________________________________________Introduction______
modules and their domain organization have a tight control over the final
product[131]. There are three major classes of PKS systems classified on the
basis of their synthesis and structural type of product. Type I PKS in bacteria are
acyl-transferases (AT) for the loading of starter, extender and intermediate acyl
units; acyl carrier proteins (ACP) which hold the growing macrolide as a thiol
reductases (ER) which catalyse the final reduction to full saturation; and finally a
PCR-based screening technique was used to screen the presence of PKS KS-
the results shows presence of KS domain which uses acyl-COA as a starter unit.
_____________________________________________________Introduction______
to the adjacent acyl carrier protein (ACP domain). These ACP’s are the second
essential domains of PKS and are analogous to the PCP domain of the NRPS
and works as a transport unit. The condensation step is similar to that of Claisen
condensation which is catalyzed by the KS-domain. Thus, it can be said that the
enumerate the exact reaction mechanism, Schwarzer & Marahiel[1] gave the
exact sequence of reactions going on after the COA-thioester moiety has been
primed.
First step in this reaction is the transfer of ketide chain to the active cystine
ketide chain. This reaction produced β-keto carboxy acid which is further
carboxy acid. These reactions are usually carried out by the ketoreductase (KR),
TE domain[137].
_____________________________________________________Introduction______
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_____________________________________________________Introduction______
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