**Arunas Krikštaponis \* and Rolandas Meškys ¯**

Department of Molecular Microbiology and Biotechnology, Institute of Biochemistry, Life Sciences Center, Vilnius University, Sauletekio al. 7, LT-10257 Vilnius, Lithuania; rolandas.meskys@bchi.vu.lt

**\*** Correspondence: arunas.krikstaponis@bchi.vu.lt; Tel.: +370-616-17187

Received: 15 September 2018; Accepted: 10 October 2018; Published: 12 October 2018

**Abstract:** A gene cluster, denoted as *hcdABC*, required for the degradation of 3-(2,4-dihydroxyphenyl)-propionic acid has been cloned from 7-hydroxycoumarin-degrading *Pseudomonas mandelii* 7HK4 (DSM 107615), and sequenced. Bioinformatic analysis shows that the operon *hcdABC* 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 confirms that this enzyme belongs to the group of *ipso*-hydroxylases. The activity of the proteins HcdB and HcdC has been analyzed by using recombinant *Escherichia coli* cells. Identification of intermediate metabolites allowed us to confirm the predicted enzyme functions and to reconstruct the catabolic pathway of 3-(2,4-dihydroxyphenyl)-propionic acid. HcdA catalyzes 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 describe a *Pseudomonas mandelii* strain 7HK4 capable of degrading 7-hydroxycoumarin via 3-(2,4-dihydroxyphenyl)-propionic acid pathway.

**Keywords:** 7-hydroxycoumarin; 3-(2,4-dihydroxyphenyl)-propionic acid; 3-(2,3,5-trihydroxyphenyl)-propionic acid; *ipso*-hydroxylase; *Pseudomonas mandelii*
