{Nonribosomal peptide synthesis and toxigenicity of cyanobacteria} ..

...stins are known to date. Microcystins are synthesised by the thiotemplate mechanism like other non-ribosomal peptides (i.e. antibiotics such as gramicidin or tyrocidin) produced by bacteria and fungi =-=[22]-=-. The large enzyme complex encoded by the mcy gene cluster is composed of peptide synthetases, polyketide synthases and tailoring functions for microcystin biosynthesis. It has a modular structure, ea...

14/12/1998 · Nonribosomal Peptide Synthesis and Toxigenicity of Cyanobacteria

In our study, we have identified three codons in the apnA A1 domain that are under positive selection. They are not located only within the putative binding pocket but are distributed over the entire A domain. In fact, the sequence variability within the binding pocket of the apnA A1 domain was indistinguishable from that affecting the entire A domain. Correspondingly, Tooming-Klunderud et al. () identified only a small number of positively selected codons that were also distributed over the entire A domains of the mcyB and mcyC genes in Anabaena, Planktothrix, and Microcystis. Recently, only two substitutions located within the mcyC A domain (1,344 bp) were observed to correlate with the replacement of Arg by Hty in position 4 of the MC molecule in Planktothrix (). We conclude that under the maximum-parsimony criterion, a rather small number of substitutions are related to the change in the substrate specificity of the ApnA A1 domain as observed in the in vitro experiments. For terpene cyclases in higher plants (Nicotiana, Hyoscyamus), detailed mutational analysis has been performed in order to characterize the catalytic landscape underlying the evolution of sesquiterpene chemical diversity (). The authors concluded that novel catalytic specificities require as little as a single nucleotide polymorphism, not necessarily located on the active-site surface of the protein. In that study, a large fraction of mutants was found to be able to produce several classes of sesquiterpenes. The authors postulated that from those promiscuous enzymes, new specific enzyme activities arise by selective pressure on the producers of these compounds. Therefore, we suggest that the ApnA A1 domains showing Arg/Tyr substrate promiscuity actually represent a transitional state in the evolutionary process of reshaping APs as enzyme inhibitors, which may result in a Tyr-specific ApnA A1 domain. If an aromatic amino acid residue in exocyclic position 1 has a selective advantage, anabaenopeptins with solely Phe, Tyr, or Trp in position 1 will become more abundant in the future. At present, only two strains (Anabaena NZ-3-1 and Nostoc PCC73102) that bear exclusively Phe in position 1 have been isolated (, ). It is notable that the Arg/Tyr dichotomy demonstrated here is also found at certain positions in other cyanobacterial metabolite families (in MCs at position 2 and in cyanopeptolins at position 2 adjacent to the 3-amino-6-hydroxy-2-piperidone [Ahp] moiety) and could have functional implications. For example, Yamaki et al. () have shown that among cyanopeptolins, the dichotomy of basic versus aromatic amino acids is decisive for either trypsin or chymotrypsin inhibition.

Nonribosomal peptide synthesis and toxigenicity of ..

21/04/1999 · Nonribosomal Peptide Synthesis and Toxigenicity of Cyanobacteria ..

Anabaenopeptins (AP) are bioactive cyclic hexapeptides synthesized nonribosomally in cyanobacteria. APs are characterized by several conserved motifs, including the ureido bond, N-methylation in position 5, and -Lys in position 2. All other positions of the AP molecule are variable, resulting in numerous structural variants. We have identified a nonribosomal peptide synthetase (NRPS) operon from Planktothrix agardhii strain CYA126/8 consisting of five genes (apnA to apnE) encoding six NRPS modules and have confirmed its role in AP synthesis by the generation of a mutant via insertional inactivation of apnC. In order to correlate the genetic diversity among adenylation domains (A domains) with AP structure variation, we sequenced the A domains of all six NRPS modules from seven Planktothrix strains differing in the production of AP congeners. It is remarkable that single strains coproduce APs bearing either of the chemically divergent amino acids Arg and Tyr in exocyclic position 1. Since the A domain of the initiation module (the ApnA A1 domain) has been proposed to activate the amino acid incorporated into exocyclic position 1, we decided to analyze this domain both biochemically and phylogenetically. Only ApnA A1 enzymes from strains producing AP molecules containing Arg or Tyr in position 1 were found to activate these two chemically divergent amino acids in vitro. Phylogenetic analysis of apn A domain sequences revealed that strains with a promiscuous ApnA A1 domain are derived from an ancestor that activates only Arg. Surprisingly, positive selection appears to affect only three codons within the apnA A1 gene, suggesting that this remarkable promiscuity has evolved from point mutations only.

Nonribosomal peptide synthetase genes occur in most ..

It involves the chemical or enzymatic treatment of a protein resulting in the formation of peptide fragments followed by separation and identification of the fragments in a reproducible manner.

Nonribosomal Peptide Synthesis and Toxigenicity of Cyanobacteria

Recently, the AP gene cluster of Anabaena strain 90, comprising seven genes (aptA to aptF), was characterized, and the biosynthetic pathway for the three AP structural variants AP A (m/z 844), AP B (m/z 837), and AP C (m/z 809), containing either Tyr, Arg, or Lys, respectively, in the exocyclic position, was proposed (). Interestingly, this AP gene cluster includes two aptA genes encoding two alternative NRPS starter bimodular proteins that putatively arose from duplication and subsequent intragenomic recombination. While the A domain of the initiation module (the AptA1 A1 domain) was not characterized biochemically, it was postulated that this module is responsible for the activation of Arg or Lys. In contrast, the A domain of the alternative starter module (the AptA2 A1 domain) was expressed and displayed high substrate selectivity for -Tyr. The authors concluded that the presence of two aptA genes enables Anabaena strain 90 to incorporate chemically distinct amino acids into the equivalent position during the biosynthesis of the three different AP variants. The same authors described the apt gene clusters of Nostoc PCC73102 and Nodularia spumigena, neither of which shows evidence of duplication of the starter module.