Extending enzyme molecular recognition with an expanded amino acid alphabet.
Extending enzyme molecular recognition with an expanded amino acid alphabet.
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通过扩展的氨基酸字母表扩展酶分子识别。
DOI：
10.1073/pnas.1616816114
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发表时间：
2017
影响因子：
11.1
通讯作者：
Windle CL
中科院分区：
文献类型：
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作者：
Windle CL
天然酶由 20 种蛋白氨基酸构建而成，然后可能需要翻译后修饰或补充辅酶或金属离子以实现催化功能。在这里，我们证明氨基酸字母表的扩展也可以使酶的特性得到扩展。化学诱变策略允许将多种非规范氨基酸系统地掺入整个活性位点，以改变酶底物特异性。具体来说，在 N-乙酰神经氨酸裂解酶 (NAL) 活性位点内的 12 个不同位置处掺入了 13 条不同的非规范侧链，并筛选了所得化学修饰酶与一系列醛底物的活性。鉴定出在 190 位含有 2,3-二羟丙基半胱氨酸的修饰酶，与野生型酶相比，其对赤藓糖与丙酮酸的醇醛缩合反应的活性显着增加。对同一位置的经典氨基酸饱和文库的动力学研究表明，20 种蛋白氨基酸中的任何一种都无法实现这种活性的增加。结构和建模研究表明，非规范侧链的独特形状和功能使得活性位点能够被重塑，从而更有效地稳定反应的过渡态。因此，利用扩展的氨基酸字母表的能力可以增强希望开发具有新催化特性的酶的蛋白质工程师的雄心。
Natural enzymes are constructed from the 20 proteogenic amino acids, which may then require posttranslational modification or the recruitment of coenzymes or metal ions to achieve catalytic function. Here, we demonstrate that expansion of the alphabet of amino acids can also enable the properties of enzymes to be extended. A chemical mutagenesis strategy allowed a wide range of noncanonical amino acids to be systematically incorporated throughout an active site to alter enzymic substrate specificity. Specifically, 13 different noncanonical side chains were incorporated at 12 different positions within the active site ofN-acetylneuraminic acid lyase (NAL), and the resulting chemically modified enzymes were screened for activity with a range of aldehyde substrates. A modified enzyme containing a 2,3-dihydroxypropyl cysteine at position 190 was identified that had significantly increased activity for the aldol reaction of erythrose with pyruvate compared with the wild-type enzyme. Kinetic investigation of a saturation library of the canonical amino acids at the same position showed that this increased activity was not achievable with any of the 20 proteogenic amino acids. Structural and modeling studies revealed that the unique shape and functionality of the noncanonical side chain enabled the active site to be remodeled to enable more efficient stabilization of the transition state of the reaction. The ability to exploit an expanded amino acid alphabet can thus heighten the ambitions of protein engineers wishing to develop enzymes with new catalytic properties.