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_____________________________________________________Introduction______

1. Introduction:

1.1 Cyanobacteria:
The cyanobacteria are an ecologically, morphologically, and physiologically

diverse group of organisms whose primary productivity contributes to the

bioenergetic foundation for higher trophic levels in marine, freshwater and

terrestrial environment. Ecologically cyanobacteria are not only capable of

modifying their habitat through fixation of atmospheric N2 but also capable of

producing biologically active natural products [3]. Cytologically they resemble

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

photosynthesis and produce oxygen, which subsequently released into the

atmosphere. Along with this beneficial part of cyanobacteria they were also

known to produce toxins. Published account of field poisoning by cyanobacteria

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

reports on such incidents were given by several authors[5-9]. Primarily, two

types of toxins, hepatotoxins and neurotoxins have been characterized from

these cyanobacteria. About 50% of Microcystis waterbloom shows hepatotoxicity

to mammals and other animals.

The chemical investigation of marine cyanobacteria for their unique natural

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

and antiviral substances[10,11]. These investigations also include several path-


_____________________________________________________Introduction______

breaking structure elucidations of these toxins. The ecological roles played by

these toxins for cyanobacteria is that of anti-grazing mechanism, to fend off

phytoplankton grazers in marine and freshwater environments[12].

In addition to toxins, cyanobacteria produce compounds of pharmaceutical

interests. The genus Nostoc GSV224 produce cryptophycin, a potent inhibitor of

microtubule assembly, which shows anticancer properties against various types

of tumors including that of multi-drug resistant tumors.

The genomic revolution has changed the face of natural product research. Over

the last two decades, more than 150 complete biosynthetic gene clusters from

bacteria, fungi and plants have been characterized[13]. Recent investigations

over the past 15 years into the genetics studies of secondary metabolites

provided an explosive impetus to the field of natural product synthesis. This

molecular prospective has focussed on some of the most pharmaceutically useful

and structurally diverse microbial metabolites belonging to the classes of

polyketide synthase (PKSs) and nonribosomal peptide synthetases (NRPs).

Because of the development of molecular approaches, there is growing trend

towards using the molecular genetics to identify biosynthetic pathways and novel

enzymes. The principal pioneer in this area was Sir David Hopwood who

identified genes encoding for the biosynthesis of actinorhodin[14]. The genes

were sequenced (a formidable task during those days) and the primary sequence

of the various proteins was established.


_____________________________________________________Introduction______

Table____: Sequenced cyanobacterial NRPS/PKS gene clusters

Gene bank Cyanobacterial strain compound


accession no

AJ269505, Anabaena Strain 90 Anabaenapeptolide[15,16]


AJ536156 Microcystin[16]

AF516145, Lyngbya majuscula strain 19L Barbamide[17], Curacin[18]


AY652953

AY522504 L. majuscula strain JHB, Jamaicamide[19],


lyngbyatoxin[20]

AF183408, Microcystis aeruginosa PCC 7806, Microcystin[21], [22]


M. aeruginosa K-139
AJ441056 Planktothrix agardhii CYA126 Microcystin[23]

AF204805, Nostoc GSV224, Nostopeptilide [24],


AY167420 Nostoc ATCC 53789 Nostocyclopeptide[25]

Table 1: Some important bioactive compounds isolated from marine and


freshwater cyanobacteria (as of January 2007)

Organism Class of Bioactivity Chemical nature Reference


compound
Microcystis Sp Lipopeptide Cytotoxic Toxin [26]

Microcystis Lipopeptide Enzyme inhibitor, cytotoxic, Aeuroginisin, [7,27,28]


aeruginosa tumor promoter, anticancer, kawaguchipeptin,
microcystin, microviridin,

Synechocystis Lipopeptide Anticancer, antiviral, Didemnin [29-32]


trididemni immunosuppressive

Lyngbya majuscula Alkaloids, anticancer, antifungal, Dolastatin, Lyngbyabellin [19,33-55]


imidazole, antimicrobial, antiviral, anti- B, microcolin A
lipopeptides inflammatory, neurotoxic, skin laxaphycins A and B,
irritant, toxin, antigrazers, homodolastatin 16
alkaline phosphatase activity, Curacin A, lyngbyabellins
antifeedant, neurotoxin, A and B, Aurilides B and
cytotoxic C, kalkitoxin
Lyngbyatoxins B and C
Lyngbya lagerheimii Sulfolipid Anti-HIV Fatty Acid (Sulfo) [56]
Oscillatoria lipopeptide Antineoplastic agent Acutiphycin and 20,21- [57]
acutissima didehydroacutiphycin
Phormidium tenue Fatty Acid Anti-HIV sulfolipid [58]
(sulfolipid)
Spirulina platensis Anti-HIV, Radical Scavenger, [59-62]
hematopoietic
Anabaena flos-aquae Lipopeptide antibiotic, anticancer alkaloide, lipopeptide [63-65]
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Aulosira fertilissima Aromatic Anticancer Aulosirazole [66]


Calothrix sp. Indoles Antimalarial, anticancer Calothrixin [67]
Cylindrospermum Alkaloid Anticancer, cytotoxic Cylindrocyclophane [68]
licheniforme
Cylindrospermopsis Alkaloid Cytotoxic Cylindrospermopsin [69]
raciborskii
Nodularia spumigena Lipopeptide Enzyme inhibitation nodularia toxin [70-72]
Nostoc sp. Amide, Anticancer, cytotoxic, Cryptophycin, [73,74]
lipopeptide antifungal, antibiotic nostophycin,
nostocyclamide,
nostocyclin
Nostoc commune Lipopeptide, Antifungal, antibiotic, Nostodione, microsporine, [75,76]
terpene, antimitotic, cytotoxic diterpenoid
oligosaccharide
Nostoc ellipsosporum Peptide and Anti-HIV, antiviral Cyanovirin [77,78]
proteins
Scytonema Lipopeptide Cytotoxic, antifungal, antiviral Halichondrin, scytophycin [79,80]
pseudohofmanni
Phormidium tenue Sulfolipids Anti-HIV, anticancer monogalactopyranosyl [58]
glycerol (MGG) and
digalactopyranosyl
glycerol (DGG)

1.2 Cyanobacterial Non-Ribosomal Peptides:

Despite increasing interest and perceived value of cyanobacterial secondary

metabolites, only few biosynthetic studies have been completed (table __)[81,82].

Consequently, very little has been known about the molecular mechanism and

biochemistry of these fascinating pathways responsible for the production of

these secondary metabolites. Some important noteworthy studies done in this

regard[18,20,21,25,83-86] but most of the studies were restricted to few

representative species, hence it has been stressed that cyanobacteria are the

most unlucky organisms having great potential and economic values but poorly

characterized[87].

Generally, it has been considered that secondary metabolites are usually

produced during stationary phase of microbes but cyanobacteria produces

bioactive peptides in all growth phases[88], but depending upon the growth

phase different metabolites may be produced in different concentration[88].

It has been a well-established fact that majority of peptide bond formation is

catalysed by ribosomes, and generally the catalytic activity of peptide bond


_____________________________________________________Introduction______

formation by nonribosomal peptide synthetases (NRPS) has been largely

overlooked. The list of molecules synthesized through NRPS is enormous[89]

such as vancomycin, which is considered as the last resort, produced by NRPS

and associate enzymes[90].

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

of repeating enzymes in a single polypeptide is similar to that of protein

machinery responsible for polyketide biosynthsis (PKS)[91]. In NRPS, one amino

acid building block is incorporated into the peptide product by each module,

hence products with 15 amino acids would be expected to be constructed by an

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

has exceptions particularly for siderophores[92,93]. Structurally, NRPS are

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

elongation module consists primarily of three basic domains: adenylation,

thiolation and condensation. The adenylation domain (A) selects a specific

amino acid and activates it as an amino acyl adenylate. The activated amino

acid is then transferred to phosphopantethiene group of the peptidyl carrier

protein (PCP) or thiolation domain (T). Condensation domain (C) catalyze the

peptide bond formation between amino acid in adjacent module. The chain is

elongated successively and released at the end by the action of thioesterase

domain (TE). Apart from these basic modules, which are ubiquitously present,
_____________________________________________________Introduction______

there also present certain tailoring/ accessory modules which certainly adds to

the structural diversity such as epimerization (E), N-methylation (MT), cyclization

(Cy), oxidoreductase (Ox), N-formylation (F), and reductase (R)[94]. These

domains helps to incorporate diverse amino acid functionality such as thiozoles,

oxazolidones, oxazoles, thiozolidines as well as other functionality like N-

methylation and D-amino acids generally not found in any other system in nature

[94].

Main Functional domains of NRPS Modules:


A large number of therapeutically useful cyclic and linear peptides of bacterial or

Figure___: Reaction catalyzed by the NRPS domains. Reaction catalyzed by principal


domains A, PCP, C & TE is given along with other auxiliary domains such as E, Cy, F,
Ox, R, and N-Mt-domains. (taken from Schwarzer et al.[1])

fungal origin are synthesized via a template-directed, nucleic-acid-independent

nonribosomal mechanism. This process is carried out by mega-enzymes called


_____________________________________________________Introduction______

nonribosomal peptide synthetases (NRPS). NRPS are organized as iterative

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

providing a linear workflow for the peptide synthesis[95,96].

A typical module comprises 1000 residues and is responsible for one reaction

cycle of selective substrate recognition and activation as adenylate, covalent

intermediate fixation in the form of enzyme-bound thioester, and peptide-bond

formation. A minimal elongation module consists of a 55 kDa adenylation (A)

domain, responsible for substrate selection and activation through ATP hydrolysis

[97,98], a 10 kDa downstream peptidyl carrier protein (PCP) domain for the

covalent fixation as a thioester [99], and a 50 kDa condensation (C) domain,

located upstream of the A domain [100] which catalyzes the peptide-bond

formation between an activated aminoacyl-bound intermediate and a peptidyl-

bound intermediate of two adjacent modules. The result is a peptide elongated

by one residue fixed to the PCP domain

and the regeneration of the PCP domain

in the preceding module. The basic set

of domains within a module can be

extended by optional modification

domains such as domain for

epimerization, N-methylation, and


Taken from Weber and Marahiel [2]
heterocyclic ring formation — which are

inserted at specific locations in the module [89]. This enlarges the broad

spectrum of possible products that results from the incorporation of non-


_____________________________________________________Introduction______

proteinogenic substrates such as carboxylic acid(for example, over 100

carboxylic acids are known as substrates). Further diversity is also achieved

through product cyclization and post-assembly modifications [101]. In its modular

organization, nonribosomal peptide synthesis resembles fatty acid synthesis

(FAS) and polyketide synthesis (PKS), which are both carried out on similarly

organized multienzyme complexes[102,103]. Furthermore, in all three cases the

cofactor used for intermediate fixation and downstream transport is a 4′-

phosphopantetheine (4′PP) moiety. This moiety is linked to a serine residue of

the PCP domain, the acyl carrier proteins (ACPs) of PKS and FAS. The cofactor

is derived from coenzyme A and post-translationally attached to the apoenzymes

of all three families by dedicated 4′PP-transferases [104].

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

substrate in NRPS adenylation domain[104]. The adenylation domain was

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

sequence homology to the tRNA. The phenylalanine-activating domain (PheA)

consists of two subdomains, a smaller C-terminal subdomain of ~100 residues

and a larger 400 residue N-terminal subdomain A (figure___). Adenylation of the

substrate amino acid (aa2) leads to aminoacyl-adenylate (aa-AMP reaction) which


_____________________________________________________Introduction______

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

(grey) which is present upstream to the A domain. The presentation of the

loaded cofactor of the PCP domain to a nucleophile acceptor position “a” and

delivery of the corresponding thioester-bound amino acid of the preceding

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

chain is attached as a thioester to the PCP domain of the following module

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

each module attached to the PCP domain.

The main difference between the ribosomal and nonribosomal systems is the

application of an accurate proofreading mechanism for ribosomal protein

synthesis but however nonribosomal synthesis shows less stringent substrate

selection and incorporation[105]. Because of the multiple carrier thiotemplate

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

observed. On the other hand in ribosomal peptide synthesis substrate selectivity


_____________________________________________________Introduction______

is relatively stringent and hence the incorporation of amino acid is highly

controlled[106]. The relaxed substrate specificity of A domain can be further

supported from the studies of Dieckmann[107] in his ATP-ppi exchange assay.

For example, BarD, it incorporate L-leucine but activates 3-chloroleucine and

valine as well[17]. The leucine specific adenylation domain of McyB of

Microcystis aerugionsa activates isoluecine and valine[108]. Similirly, the first A

domain of NosA activates Val, Ile and Leu when expressed in E. coli, but Leu is

not present in nostopeptolide[24]. In cyanobacteria, as many as 200 adenylation

domains have been identified so far. They are generally present in NRPS

system. Upon alignment, 10 core motifs (A1-A10) are highly conserved in

cyanobacterial NRPS systems which are also found in fungal system[109].

Peptidyl Carrier Protein(PCP) / Thiolation Domain (T):

This is the second domain generally found immediately after the A domain. The

key role of these domains is in the transport of intermediates, which require

specific interactions with the activating A domain and the corresponding C

domain for aminoacyl and peptidyl elongation cycle. These domains also work in

collaboration with other auxiliary domains for intermediate modifications. This

thiolation domain require interactions with epimerization domain,

methyltransferase domain, oxidation domain, reduction domain, or with

thioesterase domain in the terminal cyclization reaction[2]. The thiolation domain

(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

binding site which has a signature sequence LGx(HD)SL[96]. Both of them

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)

consists of approximately 80 residues and are composed of a distorted

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

junction between loop and the second helix.

Serine Residue Cofactor


binding pocket

Fig_____: Similarity of PCP Domains to Acyl Carrier Proteins:

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].

Condensation Domain (C):

This domain is the third domain present in the NRPS system. It catalyze the

elongation reaction of peptidyl chain which is tethered to the phosphopantetheinyl

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

(Condensation—Adenylation—Thiolation). Thus it can be said that the C

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

of C domains found in bacterial peptide synthetase system corresponds with the

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

subtypes of the C domain: i) A LCL domain which catalyzes peptide bond

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

a β-hydroxyl fatty acid). iv) Heterocyclization (Cyc) domains catalyze both

peptide bond formation and subsequent cyclization of cysteine, serine or

threonine residues. v) homologous Epimerization (E) domain flips the chirality of

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______

Thioesterase domain (TE):

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

bacteria and fungi for the synthesis of tripeptide, bacitracin, enterobactin,

gramicidin, pyoverdine, surfactin and tyrocidine[96]. In cyanobacteria, it is also

involved in the formation of Anabaenapeptolide[15,16], Microcystin[16,21-23],

Barbamide[17], Curacin[18], Nostopeptilide [24], Nostocyclopeptide[25]. Due to

its strategic location, it can be said that the TE domain might involved in

hydrolytic cleavage of the linear peptide product, i.e., termination of nonribosomal

peptide biosynthesis. The TE domain generally has a core motif of GxSxG which

is also found in acyltransferase domain of polyketide synthase. A recent

mutation study of the conserved serine residue of the signature sequence

(GxSxG) to alanine and deletion study of the entire TE domain of ACV-

synthetase of Penicillium chrysogenum to analyze the role of TE domain in

nonribosomal peptide synthetases revealed that there is drastic reduction in the

product formation in both cases which clearly underlines the importance of TE

domain[115]. Gene products of TE domains are about 220-340 amino acid

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

plays an important role in the biosynthesis of peptides in the nonribosomal

peptide synthetases system.

Other modifying domains:

Nonribosomal peptide synthetases can also carry out an array of modification

reactions N-acylation, N-methylation and epimerization. These modifying


_____________________________________________________Introduction______

domains in the nonribosomal peptide synthetases dramatically increase the

versatility and biological activity of nonribosomally synthesized peptides[2].

A) Epimerization domains:

Epimerization domains generally resembles that of condensation domains but

they have slightly different signature sequence[89]. Their main function is to

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.

B) Formyl transferase domain:

This formylatiaon domain was first identified by Rouhiainen [15] in the

anabaenopeptilide biosynthetic gene cluster. The N-terminal region shows

homology to the co-substrate formyl tetrahydrofolate-dependent methionyl-tRNA

formyltransferase. They generally shows similirities to condensation domains

and they are usually linked to the first A domain.

C) N-methylation domain:

The N-methyl transferase domain is involved in the N-methylated peptide bond

formation of the primed amino acid. This was first found in the fungal system

enniatin synthetase gene[116]. Generally N-met transferase genes are

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-

methionine (SAM)-dependent methyltransferease. Because of its insertion

between A8-A9, N-methylation function can be gained or lose by domain

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

epothilone[118] (EpoB), myxothiazol[119] (MtaC & MtaD). In epothilone

biosynthesis, this domain is involve in the oxidation of methylthiozolinyl to

methylthiazolcarboxy intermediate[120]. In case of barbamide biosynthesis gene

cluster, no oxidation domain is found in A-domain of BarG, but it has been

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

to the nucleoside-diphosphate-sugar epimerase, flavonol reductase and NADPH

dependent enzymes. In nostocyclopeptide, the final peptidyl intermediate is

reduced to the linear aldehyde cyclization to form a stable imine bond[25].

Substrate specificity of NRPS:

NRPS systems shows a moderately relaxed substrate specificity so as to allow

incorporation of more than one amino acid which is greatly responsible for the

formation of various final products in vivo (for example, tyrocidine

biosynthesis[96]. However, some positions of a particular peptide are

significantly more resistant to replacement than others, reflecting the importance

of the residues in these positions for the function of the product. The A domain

was shown to play an important role in selecting the amino acid


_____________________________________________________Introduction______

substrate[105,107]. A deep insight into the substrate binding was revealed when

the structure of the A domain of gramicidin S synthetase 1 (GrsA), complexed

with phenylalanine and adenosine monophosphate (AMP), was determined by

crystallization[121]. By comparing the sequence of the phenylalanine-binding

pocket with the adenylation domain sequences in the databases, Stachelhaus

[105] presented the selectivity-conferring code (or specificity code) of 10 amino

acids for adenylation domains. He also provided general rules for inferring the

substrate specificity tested these rules by mutations[105,113] using information

on the crystal structure of GrsA to develop a computer method for finding

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

and barG for module 2 in an amino acid-dependent ATP-pyrophosphate

exchange experiment supports the conclusion that barbamide is synthesized

from acetate, L-phenylalanine, L-cysteine and L-leucine with trichloroleucine

as a direct precursor by a mixed polyketide synthase/non-ribosomal

polypeptide synthetase, thus confirming the moderately relaxed substrate

specificity.

Colinearity between peptide synthetase and their products:

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

number of residues in the finished peptide [96,122]. Consequently, it is possible

by analysing the sequence of the NRPS genes to determine the composition of

the peptide, provided the substrate specificities of the adenylation domains are

known. In may cases, which amino acid is activated by an adenylation domain

can be deduced from the gene sequence. This is made possible by comparing
_____________________________________________________Introduction______

the so-called selectivity-conferring code of the adenylation domain with the

known precedents, as described by [105,123]. The reverse is also valid: based

on structural information the genes of a particular synthetase can be identified

from a strain that produces more than one nonribosomal peptide. Currently,

several nonlinear NRPSs are known, including the synthetases of syringomycin

[124]), yersiniabactin [125], mycobactin [126] and bleomycin [127]. Some

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

the synthetases of enterobactin from Escherichia coli [128] and of gramicidin S

from Brevibacillus brevis [129]. The activities and number of modules correspond

only to a single set of the repetitive structure of the product

Modular structure of polyketide synthase (PKS):

Fig___: Core set of elongation domain


showing Apo proteins (OH group
attached) which are unable to participate
in chain elongation. Apo proteins are
post-translationally modified with
pohsphopentathein arm in presence of
PPTase for priming and are ready for
chain elongation. (taken from Keating &
Walsh [130] )

Polyketides (PKS) are large multifunctional protein complexes which catalyze the

gradual condensation of simple building blocks. Essentially, PKS are large

modular organization and each module carries all essential information for the

recognition, activation and modification of one substrate in the form of COA

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

multienzyme complexes organized into linear modules and each module is

responsible for a single specific chain elongation process and posttranstional

modification of resulting compound.

Core PKS domains:


Natural product biosynthesis by type I PKS proceeds in a linear stepwise fashion

which begins with a loading unit. Component domains of polyketides consist of

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

ester; b-keto-acyl synthases (KS) which catalyse chain extension; b-keto

reductases (KR) responsible for the first reduction to an alochol functionality;

dehydratases (DH)which eliminate water to give an unsaturated thiolester; enoyl

reductases (ER) which catalyse the final reduction to full saturation; and finally a

thiolesterase (TE) to catalyse macrolide release and cyclisation. For

identification of the gene clusters involved in the biosynthesis of various

cyanobacterial secondary metabolites molecular approaches have been used by

several workers to elucidate the operon organization. For example, lyngbyatoxin,

curacin A, jamaicamides and barbamide from Lyngbya majuscule [17-20],

microcystin from Microcystis aeruginosa [22,23,86,108,132-134],

anabaenopeptilide from Anabaena flos-aquae [15]. Neilan et al. showed the

presence of type I PKS domains in several cyanobacteria[135,136]. A genetic

PCR-based screening technique was used to screen the presence of PKS KS-

domain in large number of laboratory and environmental samples. Analysis of

the results shows presence of KS domain which uses acyl-COA as a starter unit.
_____________________________________________________Introduction______

Subsequently, in another study Ehrenreich et al. [83], in a combined NRPS-PKS

study reported presence of NRPS A-domain and PKS KS domain in 20 marine

and freshwater cyanobacteria.

Minimally, synthesis of polyketides requires three PKS domains. The acyl

transferease (AT-domain) is responsible for the selection of substrate and is

generally similar to that of A domain of NRPS. The substrate is generally malonyl

coenzyme A thioesterase. This primed COA-thioester moiety is then transferred

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

KS domain is similar to that of Condensation (C) domain of NRPS (Fig__:). To

enumerate the exact reaction mechanism, Schwarzer & Marahiel[1] gave the

exact sequence of reactions going on after the COA-thioester moiety has been

primed.

Fig___: Reaction catalyzed by NRPS and PKS domains[1]

First step in this reaction is the transfer of ketide chain to the active cystine

residue of the KS-domain. The primed (ACP-bound) malonate is further

decarboxylated, releasing a free nucleophile which is further condensed with the

ketide chain. This reaction produced β-keto carboxy acid which is further

gradually reduced by the auxiliary domains to produce either an intermediate like

β-hydroxy carboxy acid and α, β-unsaturated ketide or a fully reduced aliphatic


_____________________________________________________Introduction______

carboxy acid. These reactions are usually carried out by the ketoreductase (KR),

dehydrogenase (DH) and enoylreductase (ER)-domains. These reactions need

NADPH as a cofactor to catalyze these reactions. The final release of the

polypeptide after completion of the elongation and reduction is catalyzed by the

TE domain[137].
_____________________________________________________Introduction______

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