Biodegradation of 7-Hydroxycoumarin in Pseudomonas mandelii 7HK4 via ipso-Hydroxylation of 3-(2,4-Dihydroxyphenyl)-propionic Acid

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.

The First Step of Biodegradation of 7-Hydroxycoumarin in Pseudomonas mandelii 7HK4 Depends on an Alcohol Dehydrogenase-Type Enzyme

“…Hence, a hydrolysis in aqueous solutions takes place, resulting in the formation of 3-(2,4dihydroxyphenyl)propionic acid. Therefore, this work in combination with the previously published data [18] allow an elucidation of a pathway of catabolism of 7-hydroxycoumarin in Pseudomonas sp. 7HK4 bacteria (Figure 9).…”

“…Thus, it was found that the transcription of the xenA38 gene was increased two-fold in the presence of 6-hydroxycoumarin and three-fold in the presence of 7-hydroxycoumarin. An expression of the xenA38 gene was also compared to the expression levels of hcdABC genes encoding a lower part of the degradation pathway of 7-hydroxycoumarin [18]. It was found that the levels of mRNA synthesis of hcdABC genes were increased 1000fold in the presence of 7-hydroxycoumarin ( Figure 3b).…”

Biodegradation of Total Petroleum Hydrocarbons in Soil: Isolation and Characterization of Bacterial Strains from Oil Contaminated Soil

“…To optimize the growth conditions for each strain in BP medium, the effects of different temperature, pH value, and incubation time on growth conditions were investigated, respectively. Different incubation temperatures (25,30,35, and 40 • C) were tested for each strain and colony-forming units (CFUs) were counted following appropriate dilution of the cultures, plating them onto fresh BP agar medium and incubating for 48 h. Similarly, the effects of different pH values (5.0, 6.0, 7.0, 8.0, and 9.0) and incubation times (3, 5, 7, and 9 days) in BP medium were also tested for each strain.…”

“…When different incubation times (3, 5, 7, and 9 d) were investigated, the colony numbers of all seven strains reached their maximum levels after 7 d incubation ( Figure 1B). In addition, we examined the effects of different incubation temperatures (25,30,35, and 40 • C) in BP medium. A 30 • C temperature was found to yield the most colony forming units for the majority of the strains ( Figure 1C), with the exception of BY6, which exhibited optimal growth at 25 • C. As shown in Figure 1D, BY1, BY2, and BY6 exhibited optimal growth at pH values around 6, whereas BY3, BY4, BY5, and BY7 yielded the most colony forming units at pH 7.…”

“…BY6 exhibited 100.0% similarity to Bacillus wiedmannii, which has been reported to be able to degrade polyethylene after 120 days incubation with a 51.53% efficiency and also exhibits biomineralization potential and hydrocarbon-degrading ability [24]. BY4 was identified as Pseudomonas mandelii, which has been reported to contain a hcdABC gene cluster that is necessary for 7-hydroxycoumarin degradation [25]. BY7 exhibited a 99.9% similarity to Pseudomonas frederiksbergensis.…”