| Abstract [eng] |
Coumarins are well known secondary metabolites widely found in various plants. However, a degradation of those compounds in the environment is not studied in detail. A soil isolate Pseudomonas mandelii 7HK4 is able to degrade 7-hydroxycoumarin (umbelliferone), but the enzymes catalyzing its transformations have not been characterized. To elucidate the pathway of catabolism of 7-hydroxycoumarin, 7-hydroxycoumarin-inducible genes hcdA, hcdB, hcdC, hcdD, hcdE, hcdF, and hcdG have been identified by an RT-qPCR analysis and protein MS-MS analysis. The DNA fragment encoding a putative alcohol dehydrogenase HcdE has been cloned, and the recombinant protein catalyses the NADPH-dependent reduction of 7-hydroxycoumarin both in vivo and in vitro. The reaction product has been isolated and characterized as 7-hydroxy-3,4-dihydrocoumarin, which proceeded through hydrolysis in aqueous solution resulting in formation of 3-(2,4-dihydroxyphenyl) propionic acid based on HPLC-MS and NMR analyses. Further, bioinformatic analysis has shown that the hcdABC genes encodes a flavin-binding hydroxylase (HcdA), an extradiol dioxygenase (HcdB), and a putative hydroxymuconic semialdehyde hydrolase (HcdC). The analysis of the recombinant HcdA activity in vitro has confirmed that this enzyme belongs to the group of ipso-hydroxylases. The activity of the proteins HcdB and HcdC has been analysed by using recombinant E. coli cells. Identification of intermediate metabolites allowed us to confirm the predicted enzyme functions and to describe the downstream catabolic pathway of 7-hydroxycoumarin via 3-(2,4-dihydroxyphenyl) propionic acid pathway. HcdA catalyses the conversion of 3-(2,4-dihydroxyphenyl) propionic acid to 3-(2,3,5-trihydroxyphenyl) propionic acid through an ipso-hydroxylation followed by an internal (1,2-C,C)-shift of the alkyl moiety. Then, in the presence of HcdB, a subsequent oxidative meta-cleavage of the aromatic ring occurs, resulting in the corresponding linear product (2E,4E)-2,4-dihydroxy-6-oxonona-2,4-dienedioic acid. Here, we have described a Pseudomonas mandelii strain 7HK4 capable of degrading 7-hydroxycoumarin via 3-(2,4-dihydroxyphenyl) propionic acid pathway. |
