Biochimie des micro-organismes, Laboratoire Charles Friedel, UMR CNRS 7223, ENSCP, 11 rue Pierre et Marie Curie, 75231 Paris Cedex 05, France.
Biochemistry. 2010 Jan 12;49(1):103-13. doi: 10.1021/bi9018785.
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.
石房蛤毒素-a 和同石房蛤毒素-a 是两种强效的蓝藻神经毒素。我们最近报道了在蓝藻中负责这些毒素生物合成的基因簇的鉴定，并提出了一个从 L-脯氨酸开始的生物合成方案，涉及三个聚酮合酶，其起始物将是与酰基辅酶 A 结合的（S）-1-吡咯啉-5-羧酸。我们现在报告了在 Oscillatoria PCC 6506 中这一生物合成的最初步骤的体外重建。我们在 PCC 6506 中鉴定出编码 Sfp 样磷酸泛酰巯基乙胺转移酶的基因，并纯化了基因产物 OsPPT，该基因产物催化磷酸泛酰巯基乙胺臂转移到 AnaD 的丝氨酸 41 位。纯的氨酰化蛋白 AnaC 将 L-脯氨酸加载到全 AnaD 上，并且是 20 种天然氨基酸中 L-脯氨酸的特异性（K(m) = 0.97 mM，k(cat) = 68 min(-1)）。在包括（S）-1-吡咯啉-5-羧酸在内的 6 种 L-脯氨酸的紧密结构类似物中，我们仅发现 3,4-脱氢-L-脯氨酸是 AnaC 的替代底物（K(m) = 1.5 mM，k(cat) = 29 min(-1)）。作为组氨酸标记蛋白纯化至均一性的假定脯氨酰-AnaD 脱氢酶 AnaB 表现出含有 FAD 的蛋白质的特征吸收光谱。如通过蛋白的胰蛋白酶消化并随后通过液相色谱与串联质谱联用所示，它将脯氨酰-AnaD 氧化为脱氢脯氨酰-AnaD。该脱氢酶的氨基酸序列与相关酶的比对表明，AnaB 属于酰基辅酶 A 脱氢酶超家族，因此可能催化硫酯结合的脯氨酸的α-β 脱氢作用，然后进行氮杂烯丙基异构化，生成（S）-吡咯啉-5-羧酸-AnaD，这是随后的聚酮合酶 AnaE 的提议起始物。
