Biology Reference
In-Depth Information
Nostoc
,
Oscillatoria
,
Plectonema
and
Pseudoanabaena
was described by Neilan
et al
. (1999) who not only
demonstrated their existence in cultures of these cyanobacteria but also from naturally occurring
bloom populations.
The precursors required for synthesis are phenylacetate, malonyl coenzyme A, S-adenyl-L-
methionine, glutamate, serine, alanine, leucine, D-methyl-isoaspartate and arginine. The gene
cluster responsible for the synthesis of MC has now been sequenced (Nishizawa
et al
., 2000; Tillett
et al
., 2000). The role of PKS has been identifi ed in the production of fatty acid side chain of Adda.
The biosynthetic pathway of MC-LR starting with the biosynthesis of Adda is presented. Tillett
et
al
. (2000) recognized two operons (from a large gene cluster) for
mcyABC
(peptide synthetase) and
mcyDE
(hybrid polyketide-peptide synthetase), as confi rmed by the cloning and sequencing of
MC synthetase gene cluster in two different strains of
M
.
aeruginosa
. The two operons recognised
are
mcyABC
and
mcyDEFGHIJ
consisting of 10 genes. The genes
mcyA
,
mcyB
and
mcyC
have
been identifi ed as responsible for activation and incorporation of fi ve amino acid constituents of
the heptapeptide (Nishizawa
et al
., 1999). Thus
mcyA1
is shown to be responsible for N-methyl-
dehydroalanine;
mcyA2
for D-alanine;
mcyB1
for Xaa (at position 2);
mcyB2
for D-erythro-β-methyl-
iso-aspartic acid;
mcyC1
for Zaa (at position 4). The second operon
mcyD
-
J
is responsible for Adda
and D-glutamate synthesis (Nishizawa
et al
., 2000; Tillett
et al
., 2000). The two operons
mcyABC
and
mcyDEFGHIJ
are transcribed in opposite directions. Apart from the six genes (
mcyA
to
mcyE
and
mcyG
) responsible for the incorporation of precursors, the rest of the four genes
were shown to
encode monofunctional proteins that possess tailoring functions. Thus these four genes, i.e.
mcyJ
,
mcyF
,
mcyI
and
mcyH
help in O-methylation, epimerization, dehydration and cellular localization,
respectively (Kaebernick
et al
., 2002).
There are three D-amino acids in MCs, i.e. D-glutamate, D-alanine and D-Me-Asp/D-aspartate
but there are only two racemase activities present in the gene cluster. An integral epimerase domain
present in the NRPS module is responsible for the incorporation of D-alanine into heptapeptide
backbone. Since an integrated epimerase domain for incorporating D-glutamate and D-MeAsp/D-
aspartate is lacking, it is suggested that these two amino acids might be converted by racemases
either before or after their incorporation. Nishizawa
et al
. (2001) showed that
mcyF
mediates synthesis
of D-glutamate since gene disruption studies abolished MC production in
M
.
aeruginosa
K-139 but
did not affect cell growth. Further,
mcyF
supported D-Glu-independent growth of an auxotroph
of
E
.
coli
for D-Glu. Thus these workers reported for the fi rst time the existence of the racemase in
prokaryotic NRPS genes. On the contrary, Sielaff
et al
. (2003) presented evidences for the synthesis
of aspartate racemases rather than glutamate racemases by
mcyF
gene and also detected a gene for
L-glutamate specifi c racemase that is located outside the
mcy
gene cluster.
It has now been possible to identify the gene clusters for heptapeptide biosynthesis in
Planktothrix
and
Anabaena
strain 90. There are certain differences in the organization of the
mcy
gene cluster
among the three MC producers (Table 4; Fig. 7). The signifi cant features of MC biosynthesis in
Planktothrix
CYA 126/8 are that (i) sequence analysis revealed
mcy
region to consist of 55-kb cluster
having 9 genes, (ii) of these, 8 genes (
mcyA
,
B
,
C
,
D
,
E
,
G
,
H
and
J
) are similar to
M
.
aeruginosa
genes
governing peptide synthetases, (iii)
mcyT
, an additional gene encoding thioesterases, is present which
is lacking in
M
.
aeruginosa
, (iv) the racemase genes
mcyF
and
mcyI
(the products of which are similar
to 3-phosphoglycerate dehydrogenase genes) present in
M
.
aeruginosa
are reported to be absent in
P
.
agardhii
, (v) while
mcyA
-
C
and
mcyD
-
J
constitute two operons that are transcribed bidirectionally
in
M
.
aeruginosa
, the whole of
mcy
gene cluster except
mcyT
represents a single operon and (vi) the
gene
mcyJ
synthesizes O-methyltransferase. The probability of lateral gene transfer (LGT) of complete
mcy
gene clusters between two different genera has been ruled out (Christiansen
et al
., 2003). In