von Bühler Clemens, Le-Huu Priska, Urlacher Vlada B
Institute of Biochemistry, Heinrich-Heine University Düsseldorf, Universitätsstrasse 1, 40225 Düsseldorf (Germany).
Chembiochem. 2013 Nov 4;14(16):2189-98. doi: 10.1002/cbic.201300271. Epub 2013 Sep 23.
Cytochrome P450 monooxygenases (P450s) are versatile enzymes with high potential for biocatalysis. The number of newly annotated P450 genes has been increasing constantly, and these thus represent a rich resource for new biocatalysts. However, the substrate scopes of newly identified P450s are often not known, and thus their exploitation is difficult. Herein we describe an approach, named "cluster screening", and its application for the primary characterization of two P450s: CYP154E1 and CYP154A8. A library comprising 51 compounds was designed and organized into nine groups according to their chemical properties. The activities of both P450s in vitro were maintained with suitable nonphysiological redox partners, and the cluster library was screened with these enzymes for product formation. From this library, 30 compounds tested positive for CYP154E1 and 23 were positive for CYP154A8. Cluster screening distinguishes subtle differences in activity and selectivity of enzymes as closely related as those of the same P450 family. For example, the alkaloid pergolide mesylate was converted by CYP154E1 (4 %) but not by CYP154A8. A building block of vitamin D3 , Grundmann's ketone, was converted by both enzymes, although conversion was higher with CYP154E1 (100 vs 53%).
细胞色素P450单加氧酶（P450s）是具有高度生物催化潜力的多功能酶。新注释的P450基因数量一直在不断增加，因此它们是新生物催化剂的丰富来源。然而，新鉴定的P450s的底物范围通常未知，因此难以对其进行开发利用。在此，我们描述了一种名为“聚类筛选”的方法及其在两种P450s（CYP154E1和CYP154A8）初步表征中的应用。设计了一个包含51种化合物的文库，并根据其化学性质将其分为九组。两种P450s在体外的活性通过合适的非生理性氧化还原伴侣得以维持，并用这些酶对聚类文库进行产物形成筛选。在该文库中，30种化合物对CYP154E1呈阳性，23种对CYP154A8呈阳性。聚类筛选能够区分同一P450家族中密切相关的酶在活性和选择性上的细微差异。例如，甲磺酸培高利特生物碱可被CYP154E1转化（4%），但不能被CYP154A8转化。维生素D3的一个结构单元，即格伦德曼酮，可被两种酶转化，不过CYP154E1的转化率更高（分别为100%和53%）。
