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

Abstract

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.

Biosynthesis of Cylindrospermopsin and 7-Epicylindrospermopsin in Oscillatoria sp. Strain PCC 6506: Identification of the cyr Gene Cluster and Toxin Analysis

“…strain PCC 6506, a benthic cyanobacterium that produces these deadly neurotoxins. We have recently described the cluster of genes responsible for the biosynthesis of these two alkaloids (24) and proposed an original biosynthetic scheme that was substantiated by in vitro experiments (23). Surprisingly, in the genome of this neurotoxic strain, we found the genes responsible for the biosynthesis of CYN, which were described previously by Mihali et al for C. raciborskii (25).…”

Insights into the Reaction Mechanism of the Prolyl–Acyl Carrier Protein Oxidase Involved in Anatoxin-a and Homoanatoxin-a Biosynthesis

“…28 Recombinant histidine-tagged, apo-AnaD, holo-AnaD, AnaC, and wild-type AnaB were obtained as previously described. 9,13 Synthesis of Deuterium-Labeled Proline. The following synthesis have been adapted from previous works.…”

“…The single peak at 15.5 min was collected, and the solution was concentrated under vacuum and analyzed by electrospray ionization mass spectrometry (ESI-MS), as already described. 13 Dithionite Reduction of AnaB and Oxidation by Air. A solution of either recombinant histidine-tagged wild-type AnaB (7.9 μM) or its E244A mutant (12.8 μM) in 20 mM Tris-HCl buffer, pH 7.7, containing 10% v/v glycerol was introduced in a cuvette fitted with a rubber septum.…”

Identification of genes for anatoxin-a biosynthesis in Cuspidothrix issatschenkoi

“…A biosynthetic pathway of the two anatoxins was also proposed (Hemscheidt et al, 1995;Mé jean et al, 2009). Briefly, proline is first activated and loaded on the acyl carrier protein AnaD by the adenylation enzyme AnaC (Mé jean et al, 2010a). The bound proline is then oxidized into dehydroproline by the oxidase AnaB using oxygen as the hydrogen acceptor (Mann et al, 2011).…”