In vitro reconstitution of the first steps of anatoxin-a biosynthesis in Oscillatoria PCC 6506: from free L-proline to acyl carrier protein bound dehydroproline.

Anatoxin-a and homoanatoxin-a are two potent cyanobacterial neurotoxins. We recently reported the identification of the gene cluster responsible for the biosynthesis of these toxins in cyanobacteria and proposed a biosynthetic scheme starting from L-proline and involving three polyketide synthases for which the starter would be (S)-1-pyrroline-5-carboxylate bound to an acyl carrier protein, AnaD. We now report the in vitro reconstitution of the first steps of this biosynthesis in Oscillatoria PCC 6506. We identified in PCC 6506 the gene coding for an Sfp-like phosphopantetheinyl transferase and purified the gene product, OsPPT, that catalyzed the transfer of the phosphopantetheinyl arm to the serine 41 of AnaD. The pure adenylation protein AnaC loaded L-proline on holo-AnaD and was specific for L-proline (K(m) = 0.97 mM, k(cat) = 68 min(-1)) among the 20 natural amino acids. Among six close structural analogues of L-proline, including (S)-1-pyrroline-5-carboxylate, we only found 3,4-dehydro-L-proline to be an alternate substrate for AnaC (K(m) = 1.5 mM, k(cat) = 29 min(-1)). The putative prolyl-AnaD dehydrogenase, AnaB, purified to homogeneity as a histidine-tagged protein, showed an absorption spectrum characteristic of FAD-containing proteins. It oxidized prolyl-AnaD to dehydroprolyl-AnaD as shown by tryptic digestion of the protein followed by liquid chromatography coupled to tandem mass spectrometry. Alignment of the amino acid sequence of this dehydrogenase with related enzymes showed that AnaB belongs to the acyl-CoA dehydrogenase superfamily and thus probably catalyzes an alpha-beta-dehydrogenation of the thioester-bound proline followed by an aza-allylic isomerization to yield (S)-pyrroline-5-carboxyl-AnaD, the proposed starter for the subsequent polyketide synthase, AnaE.

Isolation and characterization of two members of the siderophore-microcin family, microcins M and H47.

In this paper we provide the first biochemical evidence of the existence of a family of structure-related antimicrobial peptides, the siderophore-microcins, in the Enterobacteriaceae family. We isolated and characterized two novel siderophore-microcins, MccM and MccH47, previously characterized through genetic studies. MccM and MccH47 were expressed from several Escherichia coli strains containing the microcin gene clusters. The spectra of their bactericidal activities were found to be restricted to some species of the Enterobacteriaceae. MccM and MccH47 were unable to inhibit the growth of strains carrying mutations in the fepA, cir, and fiu genes, which showed the requirement of the iron-catecholate receptors for their recognition. The MccM and MccH47 peptide moieties contain 77 and 60 residues, respectively, and are derived from the microcin precursors McmA and MchB, respectively. In addition, both peptides carried a C-terminal posttranslational modification containing a salmochelin-like siderophore moiety also found in MccE492 (X. Thomas et al., J. Biol. Chem., 279:28233-28242, 2004). Interestingly, when MccM was isolated from E. coli Nissle 1917, which lacks the two genes necessary for modification biosynthesis, it was devoid of posttranslational modification. Those two genes could be complemented by their homologues from the MccH47 gene cluster, thereby showing their functional interchangeability between at least two members of the siderophore-microcin family. Finally, from the sequence analysis of the MccE492 gene cluster, we hypothesized the existence of an additional member of the siderophore-microcin family. Therefore, we propose that the siderophore-microcin family contains five representatives.

Evidence that biosynthesis of the neurotoxic alkaloids anatoxin-a and homoanatoxin-a in the cyanobacterium Oscillatoria PCC 6506 occurs on a modular polyketide synthase initiated by L-proline.

Anatoxin-a and homoanatoxin-a are potent neurotoxins produced by cyanobacteria such as Oscillatoria PCC 6506. Sequencing of the genome of this strain is underway, and we have identified a 29 kb DNA fragment containing a sequence called ks2 that we previously showed to be specific to Oscillatoria cyanobacteria producing anatoxin-a and homoanatoxin-a. Bioinformatic analysis of this 29 kb fragment revealed a cluster of genes, which were annotated. The function assigned to the products of eight contiguous genes, from anaA to anaH, provides a clue to the biosynthesis of anatoxin-a and homoanatoxin-a. Proline is first loaded on an acyl carrier protein and its five-membered cycle oxidized to the pyrroline oxidation state. This activated ring is then successively loaded on three polyketide synthase modules for elongation, reduction, cyclization, and methylation. The final step is the hydrolysis of the thioester with subsequent decarboxylation. GC-MS and NMR analyses of homoanatoxin-a produced by PCC 6506 using labeled precursors confirm that proline is very likely the starter of these polyketide synthases. Using specific PCR amplifications, we have also shown that the anaC, anaE, anaF, and anaG genes are always present in the genome of cyanobacteria producing anatoxin-a and homoanatoxin-a and absent in nonproducing strains. Histidine-tagged AnaC was purified to homogeneity and showed to catalyze the loading of proline on purified histidine-tagged AnaD that had been previously transformed into its holo form using the Bacillus subtilis Sfp phosphopantetheinyl transferase. All of these data provide strong evidence that we have successfully identified the gene cluster responsible for the production of anatoxin-a and homoanatoxin-a in Oscillatoria PCC 6506.

Insight into siderophore-carrying peptide biosynthesis: enterobactin is a precursor for microcin E492 posttranslational modification.

Microcin E492-producing bacteria secrete both unmodified and posttranslationally modified microcins. The modification consists of a C-glucosylated linear trimer of N-(2,3-dihydroxybenzoyl)-l-serine, a catecholate siderophore related to salmochelins and enterobactin. We show here that repression of enterobactin biosynthesis inhibits the acquisition of microcin E492 posttranslational modification, as monitored by high-performance liquid chromatography and mass spectrometry. Furthermore, exogenous enterobactin restored the production of posttranslationally modified microcin in a bacterial strain deficient in enterobactin synthesis. We thus concluded that enterobactin serves as a precursor for the synthesis of the posttranslationally modified microcin and that the unmodified microcin is an incompletely processed form of mature microcin E492. Gene disruption experiments showed that MceC and MceD, two enzymes encoded by the mceABCDEFGHIJ gene cluster, are involved in the synthesis of the microcin E492 posttranslational modification, as followed by mass spectrometry. Genes homologous to iroB and iroD, required for the conversion (linearization and C-glycosylation) of enterobactin into salmochelins, efficiently complemented mceC and mceD, respectively. Based on our results, a model is proposed for the biosynthesis of the mature siderophore-carrying peptide.