A patellamide-like biosynthesis of microcyclamides

Identification of the microcyclamide biosynthesis genes in M. aeruginosa NIES298 re-vealed a ribosomal assembly line similar to those from the patellamides. The gene cluster comprises 13 kb and contains nine genes, seven of which are orthologues to patellamide genes.

Unfortunately, heterologous expression of microcyclamide in E. coli failed. Although shown for patellamides (Schmidt et al., 2005), no peptides could be detected in cell pellets or supernatants of E. coli cells harbouring the mca fosmid. Since M. aeruginosa NIES298 is not genetically manipulable, possibly due to restriction endonucleases (Frangeul et al., 2008; Takahashi et al., 1996), expression in E. coli would be an important base for muta-tional studies of the biosynthesis pathway. Maybe the absence of an associated methyl-transferase in the used E. coli strain or the presence of the double glycine motif in the leader peptide hindered the production of microcyclamide. The second hypothesis is sup-ported by observations made during the expression of microviridins in E. coli. Heterolo-gous expression from the fosmids worked only for microviridin B and J from Microcystis strains, the corresponding Anabaena microviridin containing a double glycine motif in the precursor peptide was not expressed in E. coli. Many different reasons could be re-sponsible for the difficulties in heterologous expression of peptides formed from precur-sors with a double glycine motif. Differences in cell wall composition could be one reason for the difficulties resulting in a loss of function of putative transporter protease domains.

ABC transporters associated to processing and export of peptide pheromones such as colicin from E. coli are usually linked to membrane fusion proteins and outer membrane proteins such as TolC (Michiels et al., 2001). Since the peptidoglycan layer of cyanobacte-ria is considerably thicker (Hoiczyk, Hansel, 2000), interactions of cyanobactecyanobacte-rial ABC transporters and transporter-associated proteins from E. coli might be impossible.

How-Due to the similarity to the biosynthesis enzymes of patellamides, an analogous pathway to form microcyclamide seems most likely. As Schmidt and colleagues already character-ised enzymes of the patellamide pathway, no further efforts in that direction regarding microcyclamides were made. Nevertheless, sequence comparisons of precursor and modifying enzymes can reveal interesting insights to biosynthetic mechanisms.

The precursor protein McaE forms the substrate for posttranslational modifications by McaA, McaD, McaF and McaG (fig.34). McaA, analogous to PatA, catalyses the proteolytic cleavage of the N-terminal recognition sequence G(L/A)EAS in the McaE precursor pep-tide (Lee et al., 2009). The product of McaA is used as substrate for McaG. As shown for PatG (Lee et al., 2009), McaG is predicted to catalyse the cleavage of the C-terminal se-quence AFD in tandem with cyclisation of the microcyclamide peptide. A one-step transamidation mechanism was proposed to explain the cyclisation reaction (Lee et al., 2009).

Although not characterised, according to the similarity to the SagC and D families of en-zymes, it is suggested that McaD is responsible for the heterocyclisation of cysteine and serine or threonine into thiazoline and oxazoline rings. The SagBCD proteins and their homologues have been shown to introduce heterocycles onto precursor peptides of a number of bacteriocins such as microcin B17 and streptolysin S (Lee et al., 2008). Espe-cially the microcin B17 biosynthesis has been subject of extensive studies and contributes to the understanding of the basic principles of heterocyclisation in small ribosomal pep-tides (Li et al., 1996; Milne et al., 1998; Sinha Roy et al., 1999). Accordingly, McbB a SagC-homologue and zinc binding protein, is responsible for the heterocyclisation reaction.

The putative docking protein McbD, a SagD homologue including an ATPase/GTPase domain, is also required for compound production, although the exact mechanism is still elusive. In microcin B17 biosynthesis, a third protein is reqired for the heteroclisation reactions, McbC. This microcin B17 oxidase is a flavoprotein and is thought to oxidise thiazolines and oxazolines to thiazoles and oxazoles. Since no such domain is detectable in the McaD protein, the mcbC-like oxidoreductase in the N terminus of the McaG pro-tein is predicted to play that role in the microcyclamide biosynthesis.

Another enzyme is suggested to participate in heterocyclisation as well. The PatF protein was shown to be essential in patellamide biosynthesis, but is absent in the trichamide gene cluster. As no oxazoline is part of the trichamide peptide, PatF was proposed to be involved in oxazoline formation (Sudek et al., 2006).

Fig. 34 Proposed pathway to microcyclamide in M. aeruginosa NIES298.

No functional role could be assigned to McaB and McaC. Both proteins have been shown to be dispensable in patellamide biosynthesis. However, homologues are present in all known patellamide-like gene clusters, suggesting a specific role in the biosynthesis of these small cyclic peptides. Furthermore, the role of the two additional ORFs remains

that could be responsible for the methylation of histidine in microcyclamide. Therefore, it remains ambiguous whether one of the uncharacterised enzymes in the gene cluster or factors encoded in trans provide the required methylation activity. The epimerisation me-chanism is still under discussion as well. It was proposed that epimerisation of single amino acids within patellamides occurs spontaneously and is interdependent with mac-rocyclisation of the linear prepeptide. This may also explain the fact that different patel-lamide and cyclic hexapeptide variants were shown to differ in the stereochemistry of in-dividual amino acids (Banker, Carmeli, 1998; Degnan et al., 1989; Ishida et al., 2000;

Jüttner et al., 2001).

Taken together, a detailed analysis of the microcyclamide gene cluster in M. aeruginosa NIES298 has revealed the expected similarity but also clear differences from the recently described patellamide and trichamide gene clusters. This study provides the first evidence for the biosynthesis of a cyclic hexapeptide from a ribosomal precursor in cyanobacteria and suggests a similar pathway for all cyclamides.