Discovery and Biocatalytic Application of a PLP-Dependent Amino Acid γ-Substitution Enzyme That Catalyzes C-C Bond Formation
Discovery and Biocatalytic Application of a PLP-Dependent Amino Acid γ-Substitution Enzyme That Catalyzes C-C Bond Formation
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DOI：
10.1021/jacs.0c03535
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发表时间：
2020-06-10
影响因子：
15
通讯作者：
Tang, Yi
中科院分区：
文献类型：
--
作者：
Chen, Mengbin;Liu, Chun-Ting;Tang, Yi
磷酸吡哆醛（PLP）依赖性酶可以催化α、β和γ位置的L-氨基酸的转化。这些酶经常参与非蛋白质氨基酸的生物合成，作为天然产物的结构单元，并且是有吸引力的生物催化剂。在这里，我们报告了一个两步酶促合成（2S，6S）-6-甲基哌啶醇1，从桔霉素的生物合成途径的发现。关键酶CndF是PLP依赖性的，催化与环状席夫碱平衡的（S）-2-氨基-6-氧代庚酸3的合成。第二种酶CndE是立体选择性亚胺还原酶，其产生1。CndF的生化表征表明，该酶进行O-乙酰基-L-高丝氨酸的γ-消除以生成乙烯基甘氨酸酮亚胺，其受到乙酰乙酸酯的亲核攻击以在3中形成新的C-gamma-C-delta键并完成γ-取代反应。CndF对不同的β-酮羧酸酯和酯表现出混杂性。使用表达CndF和CndE的曲霉属菌株，进料各种烷基-β-酮酯导致6-取代的L-哌啶酸酯的生物合成。CndF的发现扩展了可由PLP依赖性酶催化的反应的库。
Pyridoxal phosphate (PLP)-dependent enzymes can catalyze transformations of L-amino acids at alpha, beta, and gamma positions. These enzymes are frequently involved in the biosynthesis of non-proteinogenic amino acids as building blocks of natural products and are attractive biocatalysts. Here, we report the discovery of a two-step enzymatic synthesis of (2S,6S)-6-methyl pipecolate 1, from the biosynthetic pathway of citrinadin. The key enzyme CndF is PLP-dependent and catalyzes the synthesis of (S)-2-amino-6-oxoheptanoate 3 that is in equilibrium with the cyclic Schiff base. The second enzyme CndE is a stereoselective imine reductase that gives 1. Biochemical characterization of CndF showed this enzyme performs gamma-elimination of O-acetyl-L-homoserine to generate the vinylglycine ketimine, which is subjected to nucleophilic attack by acetoacetate to form the new C-gamma-C-delta bond in 3 and complete the gamma-substitution reaction. CndF displays promiscuity toward different beta-keto carboxylate and esters. With use of an Aspergillus strain expressing CndF and CndE, feeding various alkyl-beta-keto esters led to the biosynthesis of 6-substituted L-pipecolates. The discovery of CndF expands the repertoire of reactions that can be catalyzed by PLP-dependent enzymes.