Department of Chemistry, University of Rochester, Rochester, New York 14627, United States.
J Am Chem Soc. 2011 Mar 16;133(10):3242-5. doi: 10.1021/ja109590h. Epub 2011 Feb 22.
Engineered P450 enzymes constitute attractive catalysts for the selective oxidation of unactivated C-H bonds in complex molecules. A current bottleneck in the use of P450 catalysis for chemical synthesis is the time and effort required to identify the P450 variant(s) with the desired level of activity and selectivity. In this report, we describe a method to map the active site configuration of engineered P450 variants in high throughput using a set of semisynthetic chromogenic probes. Through analysis of the resulting 'fingerprints', reliable predictions can be made regarding the reactivity of these enzymes toward complex substrates structurally related to the fingerprint probes. In addition, fingerprint analysis offers a convenient and time-effective means to assess the regioselectivity properties of the fingerprinted P450s. The described approach can represent a valuable tool to expedite the discovery of P450 oxidation catalysts for the functionalization of relevant natural products such as members of the terpene family.
工程 P450 酶是用于复杂分子中未活化 C-H 键选择性氧化的有吸引力的催化剂。目前，P450 催化在化学合成中的应用存在一个瓶颈，即需要花费时间和精力来确定具有所需活性和选择性的 P450 变体。在本报告中，我们描述了一种使用一组半合成显色探针在高通量下绘制工程 P450 变体活性位点构象的方法。通过对所得“指纹”的分析，可以可靠地预测这些酶对与指纹探针结构相关的复杂底物的反应性。此外，指纹分析提供了一种方便且有效的方法来评估指纹 P450 的区域选择性。所描述的方法可以成为加速发现 P450 氧化催化剂的有价值的工具，用于功能化相关的天然产物，如萜烯家族成员。
