Unlocking Reactivity of TrpB: A General Biocatalytic Platform for Synthesis of Tryptophan Analogues.
Unlocking Reactivity of TrpB: A General Biocatalytic Platform for Synthesis of Tryptophan Analogues.
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DOI：
10.1021/jacs.7b05007
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发表时间：
2017-08-09
影响因子：
15
通讯作者：
Arnold FH
中科院分区：
文献类型：
--
作者：
Romney DK;Murciano-Calles J;Wehrmüller JE;Arnold FH
氨基酸色氨酸（Trp）的衍生物作为化学和生物合成具有广泛生物学特性的复杂分子的前体。色氨酸类似物也是有价值的药物化学和化学生物学的工具作为积木。虽然Trp类似物的对映选择性合成通常是冗长的，并且需要使用保护基团，但酶具有以更少的步骤合成此类产物的潜力，并且具有原始的化学和立体选择性，这是生物催化的标志。酶TrpB是特别有吸引力的，因为它可以直接从丝氨酸（Ser）和相应的吲哚类似物形成Trp类似物。然而，许多潜在的有用的底物，包括庞大的或缺电子的吲哚，是很难接受的。我们已经将定向进化应用于来自激烈火球菌和海栖热袍菌的TrpB，以产生用于合成先前难处理的Trp类似物的一套催化剂。对于最具挑战性的底物，如硝基吲哚，提高活性的关键在于突变一个普遍保守和机械上重要的残基，E104。新的催化剂以高水平表达（>200 mg/L的大肠杆菌培养物），并且可以通过热处理纯化;它们可以在高达75 °C下操作（其中溶解度增强），并且可以使用Ser和容易获得的吲哚类似物作为起始材料合成在4-、5-、6-和7-位取代的对映体纯的Trp类似物。光谱分析表明，许多激活突变抑制活性亲电中间体氨基丙烯酸酯的分解，这有助于解锁TrpB的合成潜力。
Derivatives of the amino acid tryptophan (Trp) serve as precursors for the chemical and biological synthesis of complex molecules with a wide range of biological properties. Trp analogues are also valuable as building blocks for medicinal chemistry and as tools for chemical biology. While the enantioselective synthesis of Trp analogues is often lengthy and requires the use of protecting groups, enzymes have the potential to synthesize such products in fewer steps and with the pristine chemo- and stereoselectivity that is a hallmark of biocatalysis. The enzyme TrpB is especially attractive because it can form Trp analogues directly from serine (Ser) and the corresponding indole analogue. However, many potentially useful substrates, including bulky or electron-deficient indoles, are poorly accepted. We have applied directed evolution to TrpB from Pyrococcus furiosus and Thermotoga maritima to generate a suite of catalysts for the synthesis of previously intractable Trp analogues. For the most challenging substrates, such as nitroindoles, the key to improving activity lay in the mutation of a universally conserved and mechanistically important residue, E104. The new catalysts express at high levels (>200 mg/L of Escherichia coli culture) and can be purified by heat treatment; they can operate up to 75 °C (where solubility is enhanced) and can synthesize enantiopure Trp analogues substituted at the 4-, 5-, 6-, and 7-positions, using Ser and readily available indole analogues as starting materials. Spectroscopic analysis shows that many of the activating mutations suppress the decomposition of the active electrophilic intermediate, an amino-acrylate, which aids in unlocking the synthetic potential of TrpB.
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