The arrangements of the gene clusters of microcystin synthetases from three species are different (Fig. ). In Anabaena
strain 90, M. aeruginosa
), and P. agardhii
) the NRPS genes mcyA
, and mcyC
have the same order, but the organization of the other genes is different. In Anabaena
strain 90 and in M. aeruginosa
genes are in two clusters, which are transcribed in opposite directions, whereas in P. agardhii
they are in one cluster transcribed in the same direction (except mcyT
, which was not found in Anabaena
). The organization of the genes from mcyD
corresponds the order in Planktothrix
with the exception that mcyF
were not found in Planktothrix
is located after mcyC
) (Fig. ). In Anabaena
strain 90 the arrangement of the genes from mcyG
and then from mcyA
is colinear with the structure of microcystin (Fig. ). Most NRPSs follow the colinearity rule, although several exceptions are known (16
). It can be speculated that the order of the microcystin synthetase genes in Anabaena
is more original than the one in Microcystis
, in which reorganizations of these genes have taken place.
Comparison of Anabaena (A), Microcystis (B), and Planktothrix (C) microcystin synthetase gene clusters. Letters refer to the corresponding mcy genes. Figure (not to scale) shows the order of genes and direction of the gene transcription.
, the order of the domains encoded by the genes in the two sets is colinear with the hypothetical sequence of the enzymatic reactions for microcystin biosynthesis (Fig. ). The progression of the biosynthetic reactions is consistent with the order of the functions encoded first by mcyG
and continuing with the activities encoded by mcyD
, and mcyC
. Phenyl acetate is the assumed starting unit in the biosynthesis of Adda (15
). It is activated by the adenylating domain identified in the N terminus of McyG and transferred onto the subsequent thiolation site. Polyketide synthesis reactions are monitored (Fig. ). All four-extension units are malonyl-CoA molecules according to the substrate specificity of the AT domains (Fig. ). In McyG there is one KS domain to catalyze the first condensation reaction between phenylacetate and malonyl-CoA. The reductive reactions needed to fashion the polyketide chain are putatively catalyzed by KR and DH domains of McyD and McyE. The KR domain of McyG is in the right position to reduce the carbonyl group of the putative starter molecule. The methyltransferase domains of McyG, McyD, and McyE (Fig. and ) are the obvious candidates to introduce three methyl groups into the carbon frame of Adda. It was recently verified with a knockout mutant (2
) that the incorporation of the fourth methyl, which is seen in the methoxy group of Adda, is catalyzed by McyJ. The AMT domain of McyE most likely adds the amino group, which participates in the final peptide bond with the arginine residue.
There are two condensation domains of peptide synthetases in McyE. The first one logically catalyzes the peptide bond between Adda and glutamate, which is activated by the adenylation domain of McyE. The signature sequence, which was also determined as DPRHSGVVG for McyE of both M. aeruginosa and P. agardhii, has no precedents in the databases (Table ). The synthetases of other peptides, which contain glutamyl residues, are known for bacitracin, fengycin, and surfactin (accession numbers AF007865, AF023464, AF087452, and D13262). In these compounds the standard α-carboxyl of glutamate is part of the peptide bond, while in microcystins it is the γ-carboxyl. This is analogous to the activation of aspartate/methylaspartate by the second adenylation domain of McyB, which results in the β-carboxyl of aspartate/methylaspartate instead of the α-carboxyl being engaged in the peptide bond. This difference must have an impact on the compositions of the glutamate and aspartate/methylaspartate binding pockets in the adenylation domains. McyA has two adenylation domains for the activation of serine and alanine, respectively. The signature sequences of these domains have models in the databases and are almost identical in Anabaena strain 90, M. aeruginosa, and P. agardhii (Table ). The dehydration of serine supposedly takes place after the activation by adenylation and is probably catalyzed by McyI, which is similar to phosphoglycerate dehydrogenases. There is only one, internal, condensation domain in McyA, which most likely links dehydroserine and d-alanine. The C-terminal condensation domain of McyE putatively catalyzes the bond between glutamate and dehydroserine. There is a methyltransferase domain in the first module of McyA for N-methylation of dehydroserine. The epimerase domain at the C terminus of McyA converts l-alanine to the d form.
Two modules of McyB and one module of McyC logically activate and then add three residues to the nascent peptide chain: l-leucine or l-arginine, methylaspartate or aspartate, and l-arginine, respectively (Fig. ). The amino acids activated by the adenylation domains of McyC and by the first module of McyB (McyB-1) vary most frequently in microcystins. M. aeruginosa PCC7806 and M. aeruginosa K-139 produce mainly MCYST-LR, and the substrate specificity conferring sequences in McyB-1 of these strains are identical with the signature sequence for leucine (Table ). M. aeruginosa UV027 and P. agardhii CYA126 produce mostly MCYST-RR, which is also produced by Anabaena strain 90 together with MCYST-LR. Their signature sequences in McyB-1 are different and have no precedents in the databases (Table ). In M. aeruginosa UV027 the specificity codes of McyB-1 and McyC are almost identical [DVWTIGAV(E/D)WTIGAVD] and match with the codes of McyC from M. aeruginosa K-139 and M. aeruginosa PCC7806, respectively (Table ). Accordingly McyB-1 of M. aeruginosa UV027 and McyC activate arginine.
There is no epimerase domain in McyB of Anabaena
strain 90 or in the other sequenced versions of McyB, though in microcystins, the aspartyl or methylaspartyl moiety is in the d
form. The epimerization in this position and in the glutamyl residue is putatively catalyzed by McyF, which is similar to aspartate racemases, and was shown by Nishizawa et al. (19
) to complement a d
-glutamate-deficient mutant of E. coli
. The C-terminal thiosterase domain of McyC, as generally in bacterial nonribosomal peptide synthesis (10
), catalyzes the final step in microcystin biosynthesis, the cyclization of the linear peptide (Fig. ). McyH is probably not needed for the synthesis of microcystins but it may participate in the transport.
The first module of mcyB
strain 90 contains a 1,617-bp fragment, which is almost identical to the corresponding part in mcyC
. This similarity suggests a duplication of the part of mcyC
, which codes for the adenylation domain, and the replacement of the corresponding section in mcyB
. The adenylation domain mainly determines the substrate specificity in NRPSs (27
). This substitution would explain why Anabaena
strain 90 produces the arginine variant of microcystin, MCYST-RR. Because Anabaena
strain 90 also produces MCYST-LR, the first module of mcyB
has a somewhat-relaxed specificity, while mcyC
is specific to arginine.
The genes coding for the microcystin synthesis in three major producers, Anabaena
, and Planktothrix
have been sequenced. There were several differences between the microcystin synthetase genes of these three producers: (i) gene order was different (Fig. ), (ii) certain genes were lacking from some producers (Fig. ), and (iii) the gene identities were rather low (Table ). This all shows that it was necessary to characterize these genes from each organism. This research has now made it possible to design primers and probes to specifically detect and identify the toxin-producing species in natural samples even when the quantities are low (31
). These early warning methods might become effective monitoring systems and valuable tools for protecting water users.