The self-sufficient P450 RhF expressed in a whole cell system selectively catalyses the 5-hydroxylation of diclofenac.
The self-sufficient P450 RhF expressed in a whole cell system selectively catalyses the 5-hydroxylation of diclofenac.
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DOI：
10.1002/biot.201600520
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发表时间：
2017
影响因子：
4.7
通讯作者：
J. Klenk;B. Nebel;Joanne L Porter;Justyna K. Kulig;S. A. Hussain;Sven M. Richter;Michele Tavanti
中科院分区：
文献类型：
--
作者：
J. Klenk;B. Nebel;Joanne L Porter;Justyna K. Kulig;S. A. Hussain;Sven M. Richter;Michele Tavanti
P450单加氧酶能够催化许多有机分子的高度区域选择性和立体选择性氧化。然而，由于P450生物催化剂的活性、表达水平和稳定性通常较低，因此扩大这种生物氧化的规模仍然具有挑战性。尽管存在这些挑战，但它们作为重组生物催化剂越来越受欢迎，特别是用于生产药物代谢物。双氯芬酸是一种广泛使用的抗炎药物，与4′和5′羟基代谢物一起在环境中持续存在。在这里，我们利用来自红球菌属的自给自足的P450 RhF （CYP116B2）在全细胞系统中可重复催化双氯芬酸高度区域选择性氧化为5-羟基双氯芬酸。该产品是人体代谢物，因此是环境和毒理学分析的重要标准。此外，获得大量的5-羟基双氯芬酸使我们能够证明有毒的喹奈亚胺产品的进一步氧化降解。我们的研究证明了通过重组全细胞生物催化生产克级人类药物代谢物的潜力。
P450 monooxygenases are able to catalyze the highly regio- and stereoselective oxidations of many organic molecules. However, the scale-up of such bio-oxidations remains challenging due to the often-low activity, level of expression and stability of P450 biocatalysts. Despite these challenges they are increasingly desirable as recombinant biocatalysts, particularly for the production of drug metabolites. Diclofenac is a widely used anti-inflammatory drug that is persistent in the environment along with the 4'- and 5-hydroxy metabolites. Here we have used the self-sufficient P450 RhF (CYP116B2) from Rhodococcus sp. in a whole cell system to reproducibly catalyze the highly regioselective oxidation of diclofenac to 5-hydroxydiclofenac. The product is a human metabolite and as such is an important standard for environmental and toxicological analysis. Furthermore, access to significant quantities of 5-hydroxydiclofenac has allowed us to demonstrate further oxidative degradation to the toxic quinoneimine product. Our studies demonstrate the potential for gram-scale production of human drug metabolites through recombinant whole cell biocatalysis.