Department of Molecular Microbiology and Biotechnology, Institute of Biochemistry, Life Sciences Center, Vilnius University, Sauletekio al. 7, LT-10257 Vilnius, Lithuania.
Department of Organic Chemistry, Center for Physical Sciences and Technology, Akademijos 7, LT-08412 Vilnius, Lithuania.
Int J Mol Sci. 2021 Feb 4;22(4):1552. doi: 10.3390/ijms22041552.
Coumarins are well known secondary metabolites widely found in various plants. However, the degradation of these compounds in the environment has not been studied in detail, and, especially, the initial stages of the catabolic pathways of coumarins are not fully understood. A soil isolate 7HK4 is able to degrade 7-hydroxycoumarin (umbelliferone) via the formation of 3-(2,4-dihydroxyphenyl)propionic acid, but the enzymes catalyzing the α-pyrone ring transformations have not been characterized. To elucidate an upper pathway of the catabolism of 7-hydroxycoumarin, 7-hydroxycoumarin-inducible genes , , , and were identified by RT-qPCR analysis. The DNA fragment encoding a putative alcohol dehydrogenase HcdE was cloned, and the recombinant protein catalyzed the NADPH-dependent reduction of 7-hydroxycoumarin both in vivo and in vitro. The reaction product was isolated and characterized as a 7-hydroxy-3,4-dihydrocoumarin based on HPLC-MS and NMR analyses. In addition, the HcdE was active towards 6,7-dihydroxycoumarin, 6-hydroxycoumarin, 6-methylcoumarin and coumarin. Thus, in contrast to the well-known fact that the ene-reductases usually participate in the reduction of the double bond, an alcohol dehydrogenase catalyzing such reaction has been identified, and, for 7HK4, 7-hydroxycoumarin degradation via a 7-hydroxy-3,4-dihydrocoumarin pathway has been proposed.
香豆素是广泛存在于各种植物中的一类熟知的次生代谢物。然而，这些化合物在环境中的降解尚未得到详细研究，特别是香豆素的分解代谢途径的初始阶段还不完全清楚。土壤分离株 7HK4 能够通过形成 3-(2,4-二羟基苯基)丙酸来降解 7-羟基香豆素（伞形酮），但催化 α-吡喃酮环转化的酶尚未得到表征。为了阐明 7-羟基香豆素分解代谢的上游途径，通过 RT-qPCR 分析鉴定了 7-羟基香豆素诱导基因 、 、 和 。克隆了编码假定的醇脱氢酶 HcdE 的 DNA 片段，重组蛋白在体内和体外均催化 NADPH 依赖性的 7-羟基香豆素还原。根据 HPLC-MS 和 NMR 分析，分离并鉴定反应产物为 7-羟基-3,4-二氢香豆素。此外，HcdE 对 6,7-二羟基香豆素、6-羟基香豆素、6-甲基香豆素和香豆素也具有活性。因此，与众所周知的烯还原酶通常参与双键还原的事实相反，已经鉴定出一种催化这种反应的醇脱氢酶，并且对于 7HK4，已经提出了通过 7-羟基-3,4-二氢香豆素途径降解 7-羟基香豆素。
