Biosynthesis of the unusual amino acid (4R)-4-[(E)-2-butenyl]-4-methyl-L-threonine of cyclosporin A: Enzymatic analysis of the reaction sequence including identification of the methylation precursor in a polyketide pathway
Biosynthesis of the unusual amino acid (4R)-4-[(E)-2-butenyl]-4-methyl-L-threonine of cyclosporin A: Enzymatic analysis of the reaction sequence including identification of the methylation precursor in a polyketide pathway
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DOI：
10.1021/bi960224n
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发表时间：
1996-06-25
期刊：
影响因子：
2.9
通讯作者：
SchneiderScherzer, E
中科院分区：
文献类型：
--
作者：
Offenzeller, M;Santer, G;SchneiderScherzer, E
3(R)-羟基-4(R)-甲基-6(E)-辛氨酸是一种特殊氨基酸(4R)-4-[(E)-2-丁烯基]-4-甲基- l-苏氨酸（Bmt）的c9 -主链，它是由乙酰辅酶a、丙二酰辅酶a、NADPH和s -腺苷蛋氨酸通过聚酮途径作为辅酶a硫酯合成的。在这里，我们提出了详细的酶的基本组装过程的研究。在将活化的构建单元连接到Bmt聚酮合成酶后，中间体在整个循环中保持酶结合。过早切断生物合成导致中间体从酶中释放出来，或者作为辅酶A硫酯，或者在反应性c8中间体的情况下，作为内酯。酶结合的3-氧-4-己烯酸，第二个延伸周期的缩合产物，可以确定为甲基引入的唯一底物。部分生物合成包括第一次延伸周期、第二次缩合反应和甲基化步骤，遵循一个过程机制。该过程部分的所有活化中间体都可以在相应的步骤中引入正确的途径，而2-甲基3-氧-4-己烯酰辅酶a及其后的所有甲基化中间体都不能再进入循环。显然，负责生物合成途径后一部分的Bmt聚酮合成酶区域是外部供应的辅酶A硫酯无法进入的。丁基辅酶a被Bmt聚酮合成酶识别的效率与巴豆酰辅酶a相当，并被加工成3-羟基-4-甲基辛烷酰辅酶a，这是天然碱性组装产物的饱和类似物，表明Bmt聚酮合成酶对起始单元的特异性较弱。
3(R)-Hydroxy-4(R)-methyl-6(E)-octenoic acid, the C9-backbone of the unusual amino acid (4R)-4-[(E)-2-butenyl]-4-methyl-L-threonine (Bmt), is biosynthesized as a coenzyme A thioester from acetyl-CoA, malonyl-CoA, NADPH, and S-adenosylmethionine via a polyketide pathway. Here we present detailed enzymatic studies about the basic assembly process. After attachment of the activated building units to Bmt polyketide synthase the intermediates remained enzyme-bound throughout the cycle. Premature cutoff of biosynthesis led to the release of the intermediates from the enzyme, either as coenzyme A thioesters or, in the case of reactive C8-intermediates, as lactones. Enzyme bound 3-oxo-4-hexenoic acid, the condensation product of the second elongation cycle, could be identified as the exclusive substrate for the introduction of the methyl group. Part of the biosynthesis including the first elongation cycle, the second condensation reaction, and the methylation step was shown to follow a processive mechanism. All activated intermediates of this processive part could be introduced into the correct pathway at the respective steps, whereas 2-methyl 3-oxo-4-hexenoyl-CoA and all following methylated intermediates were not able to enter the cycle any more. Obviously, the region of Bmt polyketide synthase responsible for this latter part of the biosynthetic pathway is inaccessible for externally supplied coenzyme A thioesters. Butyryl-CoA was recognized by Bmt polyketide synthase with an efficiency comparable to that of crotonyl-CoA and processed to 3-hydroxy-4-methyloctanoyl-CoA, the saturated analog of the natural basic assembly product, indicating a relaxed specificity of Bmt polyketide synthase with respect to the starter unit.