Department of Pharmaceutical Biology, Groningen Research Institute of Pharmacy, University of Groningen, Antonius Deusinglaan 1, 9713 AV Groningen, The Netherlands.
Chembiochem. 2011 Mar 7;12(4):602-9. doi: 10.1002/cbic.201000633. Epub 2011 Feb 3.
The enzyme 4-oxalocrotonate tautomerase (4-OT) is part of a catabolic pathway for aromatic hydrocarbons in Pseudomonas putida mt-2, where it catalyzes the conversion of 2-hydroxy-2,4-hexadienedioate(1) to 2-oxo-3-hexenedioate(2). 4-OT is a member of the tautomerase superfamily, a group of homologous proteins that are characterized by a β-α-β structural fold and a catalytic amino-terminal proline. In the mechanism of 4-OT, Pro1 is a general base that abstracts the 2-hydroxyl proton of 1 for delivery to the C-5 position to yield 2. Here, 4-OT was explored for nucleophilic catalysis based on the mechanistic reasoning that its Pro1 residue has the correct protonation state (pK(a) ∼6.4) to be able to act as a nucleophile at pH 7.3. By using inhibition studies and mass spectrometry experiments it was first demonstrated that 4-OT can use Pro1 as a nucleophile to form an imine/enamine with various aldehyde and ketone compounds. The chemical potential of the smallest enamine (generated from acetaldehyde) was then explored for further reactions by using a small set of selected electrophiles. This systematic screening approach led to the discovery of a new promiscuous activity in wild-type 4-OT: the enzyme catalyzes the aldol condensation of acetaldehyde with benzaldehyde to form cinnamaldehyde. This low-level aldolase activity can be improved 16-fold with a single point mutation (L8R) in 4-OT's active site. The proposed mechanism of the reaction mimicks that used by natural class-I aldolases and designed catalytic aldolase antibodies. An important difference, however, is that these natural and designed aldolases use the primary amine of a lysine residue to form enamines with carbonyl substrates, whereas 4-OT uses the secondary amine of an active-site proline as the nucleophile catalyst. Further systematic screening of 4-OT and related proline-based biocatalysts might prove to be a useful approach to discover new promiscuous carbonyl transformation activities that could be exploited to develop new biocatalysts for carbon-carbon bond formation.
4-氧代戊烯酸-2,3-脱水酶（4-OT）是恶臭假单胞菌 mt-2 芳香族碳氢化合物分解途径的一部分，它可催化 2-羟基-2,4-己二烯二酸（1）转化为 2-氧代-3-己烯二酸（2）。4-OT 属于 tautomerase 超家族的一员，该蛋白家族具有 β-α-β 结构折叠和催化的氨基末端脯氨酸。在 4-OT 的机制中，Pro1 是一个广义碱，可从 1 的 2-羟基中抽取质子，转移到 C-5 位，从而生成 2。基于其 Pro1 残基具有正确的质子化状态（pK(a)∼6.4），可在 pH 7.3 下作为亲核试剂的机理推理，在此对 4-OT 的亲核催化作用进行了探索。通过抑制研究和质谱实验，首次证明 4-OT 可以利用 Pro1 作为亲核试剂，与各种醛酮化合物形成亚胺/烯胺。然后，使用一组选定的亲电试剂，探索了最小烯胺（由乙醛生成）的化学势能在进一步反应中的应用。这种系统的筛选方法导致在野生型 4-OT 中发现了一种新的混杂活性：该酶催化乙醛与苯甲醛的醛醇缩合反应，形成肉桂醛。4-OT 活性位点的单点突变（L8R）可将该酶的低水平醛醇酶活性提高 16 倍。该反应的提议机制类似于天然 I 类醛醇酶和设计的催化醛醇酶抗体的机制。然而，一个重要的区别是，这些天然和设计的醛醇酶使用赖氨酸残基的伯胺与羰基底物形成烯胺，而 4-OT 则使用活性位点脯氨酸的仲胺作为亲核催化剂。对 4-OT 和相关脯氨酸基生物催化剂的进一步系统筛选可能被证明是一种有用的方法，可以发现新的混杂羰基转化活性，从而开发用于形成碳-碳键的新生物催化剂。
