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

Article abstract of DOI:10.3390/molecules23102613

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

Full text of DOI:10.3390/molecules23102613

molecules
Article
Biodegradation of 7-Hydroxycoumarin in
Pseudomonas mandelii 7HK4 via ipso-Hydroxylation
of 3-(2,4-Dihydroxyphenyl)-propionic Acid
Ar u¯ nas 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
ꢀ
ꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀ
ꢁꢂꢃꢄꢅꢆ
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
1
. Introduction
The compound 7-hydroxycoumarin (
plant-derived secondary metabolites. It is a parent compound of other, more complex natural
furanocoumarins and pyranocoumarins in higher plants [ ]. In the case of plant damage, plants
produce a high diversity of natural coumarins as a defense mechanism against insect herbivores as
well as fungal and microbial pathogens [ ]. For example, simple hydroxycoumarins have antibacterial
activity against Ralstonia solanacearum, Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus,
and Pseudomonas aeruginosa [ ]. Despite the toxic effects of coumarins, it has been shown that
5
), also known as umbelliferone, is one of the most abundant
1–3
4
4–8
microorganisms evolve to gain the ability to metabolize such compounds.
It has been shown previously that a number of soil microorganisms, such as Pseudomonas, Arthrobacter,
Aspergillus, Penicillium, and Fusarium spp. can grow on coumarin (
The key intermediate during coumarin catabolism in bacteria is 3-(2-hydroxyphenyl)-propionic acid
) [10 11 16]. The bioconversion of coumarin to 3-(2-hydroxyphenyl)-propionic acid can be achieved in
two different metabolic pathways, as shown in Figure 1A. Bacteria belonging to the Arthrobacter genus
enzymatically hydrolyze the lactone moiety to give 3-(2-hydroxyphenyl)-2-propenoic acid ( ), and then
1) as a sole source of carbon [9–16].
(4
, ,
2
Molecules 2018, 23, 2613; doi:10.3390/molecules23102613
www.mdpi.com/journal/molecules

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reduce a double bond by using a NADH-dependent enzyme to yield 3-(2-hydroxyphenyl)-propionic
acid [11]. In the case of Pseudomonas sp. 30-1 and Aspergillus niger ATCC 11394 cells, coumarin
is initially reduced to dihydrocoumarin (
enzymatically hydrolyzed [ 10 14]. Arthrobacter and Pseudomonas species are capable of oxidizing
-(2-hydroxyphenyl)-propionic acid to 3-(2,3-dihydroxyphenyl)-propionic acid by using specific
3) by a NADH-dependent oxidoreductase and only then
9, ,
3
flavin-binding aromatic hydroxylases [16]. However, no data are available on further conversions
of 3-(2,3-dihydroxyphenyl)-propionic acid in these bacteria. Also, no microorganisms or enzymes
implicated in the metabolism of any hydroxycoumarin have been identified to date.
Figure 1.
and Aspergillus spp. (c, d). (
sp. 7HK4 bacteria. —coumarin;
—3-(2-hydroxyphenyl)-propionic acid;
acid; —putative coumarin
(
A
) Coumarin metabolic routes in Arthrobacter spp. (a, b), as well as Pseudomonas
) Proposed metabolic pathway of 7-hydroxycoumarin in Pseudomonas
—3-(2-hydroxyphenyl)-2-propenoic acid; —dihydrocoumarin;
—7-hydroxycoumarin; —3-(2,4-dihydroxyphenyl)-propionic
hydrolase; —NADH:o-coumarate oxidoreductase;
B
1
2
3
4
5
6
a
b
c—dihydrocoumarin:NAD[NADP] oxidoreductase; d—putative dihydrocoumarin hydrolase.
Here we describe a Pseudomonas sp. 7HK4 strain capable of utilizing 7-hydroxycoumarin
as the sole carbon and energy source, and a catabolic pathway of 7-hydroxycoumarin in these
bacteria. Analysis of 7-hydroxycoumarin-inducible proteins led to the identification of the genome
locus encoding 7-hydroxycoumarin catabolic proteins. The corresponding genes were cloned and
heterologously expressed in E. coli system. The functions of the recombinant proteins were determined
and enzyme activities towards various substrates were evaluated. We show that Pseudomonas sp.
7HK4 encodes a distinct 7-hydroxycoumarin metabolic route, which utilizes a flavin monooxygenase
responsible for ipso-hydroxylation of 3-(2,4-dihydroxyphenyl)-propionic acid (6).
2
. Results
2
.1. Screening and Identification of 7-Hydroxycoumarin-Degrading Microorganisms
By the means of enrichment culture using various coumarin derivatives, an aerobic strain
HK4 degrading 7-hydroxycoumarin was isolated from the garden soil in Lithuania (DSMZ accession
7
number DSM 107615). This bacterium was tested for its ability to grow on several coumarin derivatives,
such as coumarin, 3-hydroxycoumarin, 4-hydroxycoumarin, 6-hydroxycoumarin, 6-methylcoumarin,
6
,7-dihydroxycoumarin, and 7-methylcoumarin, as the sole carbon and energy source in a minimal
salt medium. However, of all the aforementioned compounds, the strain 7HK4 was able to utilize
-hydroxycoumarin only. The strain utilized glucose, which was used as a control substrate in
7
whole-cell reactions. Data on biochemical analysis of this strain are given in the Supplementary
Material. The nucleotide sequence of 16S rRNA gene was determined by sequencing of the cloned
DNA fragment, which was obtained by PCR amplification. The strain 7HK4 showed the highest

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16S rDNA sequence similarity to that from Pseudomonas genus and was similar to 16S rDNA from
Pseudomonas mandelii species according to the phylogenetic analysis, as shown in Figure S1 in the
Supplementary Material.
2
.2. Bioconversion Experiments by Using Whole Cells of Pseudomonas sp. 7HK4
Time course experiments using whole cells of Pseudomonas sp. 7HK4 pre-grown in the
presence of 7-hydroxycoumarin showed that the cells converted coumarin, 6-hydroxycoumarin,
and 6,7-dihydroxycoumarin in addition to 7-hydroxycoumarin, according to the changes in the
UV-VIS spectra, as shown in Figure S2 in the Supplementary Material. UV absorption maxima
were dropping down over time, although the biotransformation rates of 6-hydroxycoumarin,
6,7-dihydroxycoumarin, and coumarin were approximately 5-fold to 10-fold lower, respectively.
After the completion of bioconversions, there were no visible spectra observed for the
residual aromatic compound in the reaction mixtures with 7-hydroxycoumarin, except for
the reactions with 6-hydroxycoumarin, 6,7-dihydroxycoumarin, and coumarin, which had
non-disappearing UV absorption maxima at 260–270 nm wavelengths. These spectra are similar
to that of 3-phenylpropionic acid (data not shown), suggesting that 7-hydroxycoumarin-induced
7
6
3
HK4 strain can only catalyze the hydrolysis and reduction of lactone moiety of coumarin,
-hydroxycoumarin, and 6,7-dihydroxycoumarin producing 3-(2-hydroxyphenyl)-propionic,
-(2,5-dihydroxyphenyl)-propionic, and 3-(2,4,5-trihydroxyphenyl)-propionic acids, respectively,
comparable to similar biotransformations in other microorganisms [9–16]. In addition, uninduced
Pseudomonas sp. 7HK4 cells grown in the presence of glucose showed delayed and slower conversion
of 7-hydroxycoumarin. The bioconversion process only started 0.5 h after the addition of substrate,
suggesting that 7-hydroxycoumarin induced its own metabolism. In addition, uninduced cells did
not catalyze any conversion or showed delayed and much slower biotransformations for coumarin,
6
,7-dihydroxycoumarin, and 6-hydroxycoumarin, respectively. This demonstrates that in Pseudomonas
sp. 7HK4 bacteria, the metabolism of 7-hydroxycoumarin is an inducible process.
It has been shown previously that 3-(2-hydroxyphenyl)-2-propenoic
-(2-hydroxyphenyl)-propionic acids, as shown in Figure 1B, are the intermediates in a known
coumarin metabolic pathway in several microorganisms [ 11 14 16]. By analogy, it was
suggested that 3-(2,4-dihydroxyphenyl)-propionic acid may be an intermediate metabolite in
-hydroxycoumarin catabolism, as shown in Figure 1B. The cells of 7HK4 strain pre-cultivated in the
and
3
9– , ,
7
presence of 7-hydroxycoumarin catalyzed no conversion of 3-(2-hydroxyphenyl)-2-propenoic
or 3-(2-hydroxyphenyl)-propionic acid, however 3-(2,4-dihydroxyphenyl)-propionic acid
was straightforwardly consumed.
The HPLC-MS analysis of the bioconversion mixtures
showed that Pseudomonas sp. 7HK4 cells cultivated in the presence of 7-hydroxycoumarin
produce 3-(2,4-dihydroxyphenyl)-propionic acid as an intermediate metabolite, as shown in
Figure 2. In 7HK4 bacteria grown on glucose, the aforementioned compound was not observed
suggesting that 3-(2,4-dihydroxyphenyl)-propionic acid is an intermediate metabolite during
7
-hydroxycoumarin degradation.

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Figure 2. Pseudomonas sp. 7HK4 bacteria were grown in the presence of 7-hydroxycoumarin,
and produced metabolites were analyzed by HPLC-MS. UV 254 nm trace of metabolites ( ). UV and MS
spectra of peaks with retention times 5.082 min ( ) and 5.759 min ( ). The negative ions [M
A
−
H]
B
,D
C
,E
−
generated are at m/z 181.00 (3-(2,4-dihydroxyphenyl)-propionic acid) and 161.05 (7-hydroxycoumarin).
2
.3. Identification of 7-Hydroxycoumarin-Inducible Proteins
To elucidate which enzymes are involved in 7-hydroxycoumarin metabolism, Pseudomonas sp.
7HK4 cells were cultivated in a minimal medium supplemented with 7-hydroxycoumarin (0.3 mM) or
glucose (0.3 mM) as the sole carbon and energy source. Several 7-hydroxycoumarin-inducible proteins
of different molecular mass were observed by using SDS-PAGE analysis of cell-free extracts from
Pseudomonas sp. 7HK4, as shown in Figure 3A.
Three bands corresponding to the inducible proteins of 23, 32, and 50 kDa, as shown in Figure 3A,
were excised from the SDS-PAGE gel and analyzed by MS-MS sequencing. The genome sequence of
strain 7HK4 was obtained via Illumina sequencing as described in Materials and Methods. For the
identification of corresponding genes of inducible proteins, the sequences of the identified peptides
were searched against the partial 7HK4 genome sequence. Thus, the genome fragment was discovered,
encoding a 31.2 kDa protein containing 16 aa-long sequence, as shown in the bolded sequence in
Figure 3A, identical to that found in 7-hydroxycoumarin-inducible ~32 kDa protein.

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Figure 3.
cultivated in the presence of glucose (lane 1) or 7-hydroxycoumarin (lane 2). M—molecular mass ladder
kDa). The arrows indicate 7-hydroxycoumarin-inducible 23, 32, and 50 kDa proteins. The peptide
(A) SDS-PAGE analysis of cell-free extracts of Pseudomonas sp. 7HK4. Bacteria were
(
sequences determined by MS-MS are given on the right. The sequence in bold was identified after
the peptide sequences obtained by MS-MS were compared with the partially sequenced genome
of Pseudomonas sp. 7HK4.
The arrows indicate open reading frames (ORFs) encoding HcdA, HcdB, and HcdC. The gene for the
-hydroxycoumarin-inducible protein of 31.2 kDa is marked by an asterisk.
(B) Organization of hcd genes in Pseudomonas sp. 7HK4 bacteria.
7
2
.4. Analysis of the Genome Locus Encoding the 7-Hydroxycoumarin-Inducible Protein
Analysis of Pseudomonas sp. 7HK4 genome sequences (35 contigs) showed that the inducible
31.2 kDa protein belongs to the fumarylacetoacetate (FAA) hydrolase family, which includes
such enzymes as 2-keto-4-pentenoate hydratase, 2-oxohepta-3-ene-1,7-dioic acid hydratase,
-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate hydrolase, or bifunctional isomerases/decarboxylases
catechol pathway), as shown in Figure S3 in the Supplementary Material. The FAA family proteins are
usually involved in the last stages of bacterial metabolism of aromatic compounds [17 19], suggesting
2
(
–
that 31.2 kDa protein from Pseudomonas sp. 7HK4 participates in the final steps of 7-hydroxycoumarin
metabolism, after oxidative cleavage of the aromatic ring.
Adjacent to the 31.2 kDa protein-encoding gene, two open reading frames (ORFs) were identified.
All three genes are arranged on the same DNA strand, and are separated by short intergenic regions,
suggesting that these genes are organized into an operon, as shown in Figure 3B. The putative
operon was designated hcdABC (
hydroxycoumarin degrading operon), where hcdC encodes the
31.2 kDa protein. A BLAST analysis of hcdA and hcdB sequences revealed that these genes encode
the putative FAD-binding hydroxylase and ring-cleavage dioxygenase, respectively. HcdA protein
was not assigned to any family, but it showed similarity to a putative 2-polyprenyl-6-methoxyphenol
hydroxylase, as shown in Figure S3 in the Supplementary Material. This type of enzyme belongs to
class A of FAD-binding monooxygenases, which are involved in bacterial degradation of aromatic
compounds [20–23]. The product of the hcdB gene belongs to the cl14632 superfamily that combines a
variety of structurally related metalloproteins, including the type I extradiol dioxygenases, as shown in
Figure S3 in the Supplementary Material. The type I extradiol dioxygenases catalyze the incorporation
of both atoms of molecular oxygen into aromatic substrates that results in the cleavage of the aromatic
rings [24,25].

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2
.5. Expression and Substrate Specificity of HcdA Hydroxylase
For further characterization of HcdA hydroxylase, the hcdA gene was amplified by PCR and
cloned into the pET21b expression vector. The sequence was confirmed by Sanger sequencing.
The recombinant C-terminally His -tagged protein was produced in Escherichia coli BL21, and purified
6
by affinity chromatography. The purified enzyme migrated as a ~62 kDa band on SDS-PAGE, as shown
in Figure S4A in the Supplementary Material, and had a bright yellow color with absorbance maxima
at 380 and 450 nm wavelengths, as shown in Figure S4B in the Supplementary Material, suggesting
that the protein contains a tightly bound flavin [26–28]. The gel-filtration showed that the purified
HcdA protein is a monomer, as shown in Figure S4C in the Supplementary Material. The specificity
for both flavin and nicotinamide cofactors was investigated. The HcdA hydroxylase was able to
utilize either NADH or NADPH, although the oxidation rates of NADPH were almost 2-fold lower.
Kinetic characterization of HcdA protein is presented in the Supplementary Material.
The activity of the HcdA enzyme was assayed in the presence of NADH cofactor
against various substrates.
presence of 3-(2,4-dihydroxyphenyl)-propionic acid.
when trans-2,4-dihydroxycinnamic acid was used as the substrate.
active towards trans-cinnamic, cis-2,4-dihydroxycinnamic, 3-(2-hydroxyphenyl)-propionic,
-(2-hydroxyphenyl)-2-propenoic, 3-(4-hydroxyphenyl)-2-propenoic, 3-(3-hydroxyphenyl)-2-propenoic,
-(2-bromophenyl)-propionic, 3-(2-nitrophenyl)-propionic, and 3-phenylpropionic acids, cinnamyl alcohol,
The highest rate of oxidation of NADH was recorded in the
A 40-fold lower rate was observed
The HcdA was not
3
3
pyrocatechol, 3-methylcatechol, 4-methylcatechol, 2-propylphenol, 2-propenylphenol, 2-ethylphenol,
o-cresol, o-tyrosine, resorcinol, 2,3-dihydroxypyridine, 2-hydroxy-4-aminopyridine, N-methyl-2-pyridone,
N-ethyl-2-pyridone, N-propyl-2-pyridone, N-butyl-2-pyridone, indoline, and indole. These data
demonstrate that the HcdA is a highly specific monooxygenase, which shows a strong preference to
3
-(2,4-dihydroxyphenyl)-propionic acid. The addition of His -tag did not affect the enzymatic activity of
6
the HcdA protein.
The product of the reaction catalyzed by the HcdA hydroxylase was analyzed by UV-VIS
absorption spectroscopy and HPLC-MS. A new UV absorption maximum was observed at
40 and 490 nm upon addition of 3-(2,4-dihydroxyphenyl)-propionic acid to the reaction mixture.
3
The consequent red coloring was observed, which indicated the presence of para- or ortho-quinone [29],
a presumed product of the autooxidation of the corresponding hydroquinone. The same coloration was
also observed in vivo when Pseudomonas sp. 7HK4 cells were grown in an excess of 7-hydroxycoumarin
and when Escherichia coli BL21 cells harboring the p4pmPmo plasmid were cultured in the presence of
3-(2,4-dihydroxyphenyl)-propionic acid. HPLC-MS analysis of the reaction mixtures of both in vitro
and in vivo bioconversions confirmed the formation of 3-(trihydroxyphenyl)-propionic acid and
−
quinone, found [M
−
H] masses were 197.00 (traces seen only in vivo) and 195.00, respectively,
as shown in Figure 4. However, the structure of the product was not confirmed at this stage by
chemical analysis since it was not possible to chromatographically separate the product from the
reaction mixture. The substrate and product have similar structures and chemical properties; therefore,
both were detected under the same HPLC-MS chromatogram peak with retention time of ~5.5 min.

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Figure 4. HPLC-MS analysis of 3-(2,4-dihydroxyphenyl)-propionic acid bioconversion mixture in vitro
.
UV 254 nm trace of 3-(2,4-dihydroxyphenyl)-propionic acid and its hydroxylated product under the
same peak with retention time 5.472 min (on the left), and MS spectrum of the dominant peak (on the
−
right). The negative ions [M
−
H] generated are at m/z 181 (3-(2,4-dihydroxyphenyl)-propionic
acid), 195 (product of 3-(trihydroxyphenyl)-propionic acid autooxidation), and 363 (dimer of
-(2,4-dihydroxyphenyl)-propionic acid).
3
2
.6. Expression and Substrate Specificity of HcdB Dioxygenase
HcdB dioxygenase was expressed from the plasmid pTHPPDO in E. coli BL21. All attempts
to purify HcdB aerobically resulted in the loss of the enzymatic activity, even in the presence of
organic solvents, such as glycerol, ethanol, and acetone that were known as stabilizers for similar
enzymes [30–32]. The addition of dithiothreitol and ferrous sulfate to the aerobically purified protein
did not restore its activity, although it had been shown to activate and/or stabilize other extradiol
dioxygenases [30–34].
Therefore, due to the highly unstable nature of the HcdB enzyme, all the activity measurements
were carried out in vivo using the whole cells of E. coli BL21 transformed with the pTHPPDO plasmid.
The bioconversion of pyrocatechol, 3-methylcatechol, 3-methoxycatechol, 4-methylcatechol, and caffeic
acids in each case produced yellow products with the absorbance maxima expected for the proximal
meta-cleavage products of these catechols, as shown by the solid line in Figure 5 [35–37]. A weak
yellow color was visible in bioconversion mixture containing 3-(2,3-dihydroxyphenyl)-propionic
acid, and no changes in color took place, but the absorbance maxima shifts were observed
0
0
0
during reactions with pyrogallol, gallacetophenone, 2 ,3 -dihydroxy-4 -methoxyaceto-phenone
hydrate, 3,4-dihydroxybenzoic acid, 2,3,4-trihydroxybenzoic acid, and 2,3,4-trihydroxy-benzophenone,
as shown in Figure S11 in the Supplementary Material. No activity was observed with
1,2,4-benzenetriol and 6,7-dihydroxycoumarin. The E. coli cells without the hcdB gene showed no
activity towards the aforementioned compounds. The expected shift of peaks of the UV-VIS spectra of
the reaction products to a shorter wavelength were observed after acidification of the reaction mixtures
containing pyrocatechol, 3-methylcatechol, 3-methoxycatechol, 4-methylcatechol, and caffeic acid,
as shown by the dashed line in Figure 5 [38,39]. These findings revealed that hcdB encodes an extradiol
dioxygenase, which can utilize a number of differently substituted catechols.
Furthermore, the HcdB dioxygenase was co-expressed with the HcdA hydroxylase in E.
coli BL21 cells. The activity of those cells towards 3-(2,4-dihydroxyphenyl)-propionic acid was
analyzed. No coloration occurred during the incubation of over 72 h, compared to the formation
of a reddish bioconversion product by E. coli cells containing the hcdA gene only. Products of
3
-(2,4-dihydroxyphenyl)-propionic acid conversion by HcdA and HcdB were analyzed by HPLC-MS.
−
Ions with masses 181.00, 195.00, 197.00 ([M
−
H] ) were not detected showing a complete

Molecules 2018, 23, 2613
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−
conversion of substrate and its hydroxylated forms. Also, none of the expected ions ([M
−
H]
+
2
3
29.00 or [M+H] 231.00) were observed for a presumed product of the oxidative cleavage of
-(trihydroxyphenyl)-propionic acid.
Figure 5. Biotransformations of pyrocatechol (A), 3-methylcatechol (B), 3-methoxycatechol (C),
4-methylcatechol (D), and caffeic acid (E) by whole cells of E. coli BL21 containing hcdB gene.
Biotransformations were carried out in 50 mM potassium phosphate buffer pH 7.5 (solid line) at
◦
3
0 C with 0.5–1 mM of substrate. Incubation time is shown in min. Dashed lines indicate peak shifts
to a shorter wavelength after acidification of reaction mixtures.
2
.7. Isolation and Identification of 3-(2,4-Dihydroxyphenyl)-propionic Acid Bioconversion Product
Due to difficulties in detecting colorless meta-cleavage product of catechol derivative, and since
no reasonable mass spectra could be registered, we decided to transform a cleavage product into
the derivative of picolinic acid by incubation with NH Cl as described in Materials and Methods.
4
The formation of a picolinic acid derivative was proven by HPLC-MS analysis, which showed the
−
formation of [M
−
H] ion 210.00 mass, as shown in Figure 6, corresponding to the addition of NH to
3
the meta-cleavage product and the loss of two H O molecules [40].
2
1
The H NMR spectrum of this derivative [
δ
7.73 (s, 1H), 6.89 (s, 1H), 2.65 (t, J = 7.4 Hz, 2H),
2.38 (t, J = 7.4 Hz, 2H)] showed a set of two aryl protons that, from the coupling pattern (singlet
+
singlet), were in meta- or para-positions to each other on the aromatic ring [41], as shown in
Figure S12 in the Supplementary Material. The appearance in the spectrum of two triplets with
chemical shifts of 2.38 and 2.65 ppm indicated the presence of four methylene protons [41]. The 13
C
NMR spectrum [
δ
181.51, 179.99, 171.01, 143.14, 136.74, 130.11, 115.22, 35.50, 24.00] showed two
3
2
sp carbons with chemical shifts of 24.00 and 35.50 ppm, and three sp carbons of carbonyl groups
with chemical shifts of 171.01, 179.99, and 181.51 ppm, respectively, as shown in Figure S13 in the
Supplementary Material. The carbonyl carbon atoms were the most strongly deshielded and their
2
resonances formed a separate region at the highest frequency. Another four sp carbon signals
were in the aromatic carbon region [41,42]. The presence of the third carbonyl group indicated
the formation of oxo-pyridine, for which six possible theoretical structures of oxo-picolinic acid
1
derivative were presumed, as shown in Figure 7. Since the H NMR spectrum showed a set of
two singlet aryl protons in meta- or para-positions to each other, only structures 7 and 9, as shown in
Figure 7, were further analyzed. Besides, pyridine aromatic carbons are usually differentiated into two
resonances at higher field (C-3/5, meta position) and three at lower field (C-2/6, ortho position; C-4,
para position), where the electron-withdrawing effect of nitrogen is effective [41,42]. The chemical shift

Molecules 2018, 23, 2613
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of 115.22 ppm showed that the analyzed compound had relatively strongly shielded unsubstituted
aromatic carbon, which should be in meta-position from nitrogen, in ortho-position from the carbonyl
group, and in meta- or para-position from the carboxyl group (C-5 atom), as shown in Figure S14 in
the Supplementary Material [43
,44]. This led to the conclusion that structure 9, as shown in Figure 7,
6-(2-carboxyethyl)-4-oxo-1,4-dihydropyridine-2-carboxylic acid, was formed during incubation of
meta-cleavage product of catechol derivative with NH Cl, as depicted in Scheme 1. These data
4
allowed the reconstruction of the consecutive oxidation of 3-(2,4-dihydroxyphenyl)-propionic
acid catalyzed by the HcdA and HcdB enzymes. Hence, 3-(2,3,5-trihydroxyphenyl)-propionic
acid (14) was the product of oxidation of 3-(2,4-dihydroxyphenyl)-propionic acid by the
HcdA hydroxylase. The molecular mass of 198.17 of 3-(2,3,5-trihydroxyphenyl)-propionic acid
and capability to form para-quinone agreed with the UV-VIS and HPLC-MS data on the
bioconversion of 3-(2,4-dihydroxyphenyl)-propionic by the HcdA hydroxylase. The formation
of 3-(2,3,5-trihydroxyphenyl)-propionic acid from 3-(2,4-dihydroxyphenyl)-propionic acid was
possible only through oxidative ipso-rearrangement, a unique reaction where ipso-hydroxylation
(13) of the 3-(2,4-dihydroxyphenyl)-propionic acid takes place with a simultaneous shift of the
propionic acid group to the vicinal position, as shown in Scheme 1 [45 48]. During the second
–
step, 3-(2,3,5-trihydroxyphenyl)-propionic acid was cleaved by HcdB extradiol dioxygenase at the
meta-position leading to the formation of (2E,4E)-2,4-dihydroxy-6-oxonona-2,4-dienedioic acid (15).
The further imine formation and tautomerization in the presence of ammonium ions [38
,49] led to
6
-(2-carboxyethyl)-4-oxo-1,4-dihydropyridine-2-carboxylic acid (19), as shown in Scheme 1.
Figure 6. HPLC-MS analysis of 3-(2,4-dihydroxyphenyl)-propionic acid bioconversion mixture in vivo
.
UV 254 nm trace of picolinic acid derivative with retention time of 4.500 min (A), UV spectrum (B) and
MS spectrum (C) of the dominant peak. The negative ions [M−H]− generated are at m/z 210.00.

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Figure 7. Suggested structures of oxo-picolinic acid derivative, formed during oxidative ring cleavage
of 3-(2,4-dihydroxyphenyl)-propionic acid and conversion of the ring fission product. Solid lines
indicate possible positions of hydroxylation by HcdA enzyme; hollow arrays indicate the probable
oxo-picolinic acid derivatives forming after hydroxylation at each position.
Scheme 1.
The proposed metabolic pathway of 7-hydroxycoumarin in Pseudomonas sp.
Incubation of the compound with NH Cl gives picolinic acid derivative.
—7-hydroxycoumarin; —3-(2,4-dihydroxyphenyl)-propionic acid;
acid; 14—3-(2,3,5-trihydroxyphenyl)-propionic
—(2E,4E)-2,4-dihydroxy-6-oxonona-2,4-dienedioic acid; 16—(E)-2-hydroxy-4-oxopent-2-enoic
acid; 17—succinic acid; 18—6-(2-carboxyethyl)-4-hydroxypicolinic acid; 19—6-(2-carboxyethyl)-4-
oxo-1,4-dihydropyridine-2-carboxylic acid; HcdA—3-(2,4-dihydroxyphenyl)-propionic acid
-monooxygenase; HcdB—3-(2,3,5-trihydroxyphenyl)-propionic acid 1,2-dioxygenase; HcdC—putative
2E,4E)-2,4-dihydroxy-6-oxonona-2,4-dienedioic acid hydrolase. The dashed arrow indicates a
hypothetical reaction.
7HK4 cells.
5
4
5
6
13—3-(1,2-dihydroxy-4-
acid;
oxocyclohexa-2,5-dienyl)-propanoic
1
5
1
(

Molecules 2018, 23, 2613
11 of 19
2
.8. Expression of the HcdC Protein
HcdC was expressed from the plasmid p2K4PH in E. coli BL21. The addition of E. coli
extracts containing the HcdC protein did not cause decolorization of the meta-cleavage products of
3
-(2,3-dihydroxyphenyl)-propionic acid, pyrocatechol, 3-methylcatechol, or 4-methylcatechol formed
+
in the presence of the HcdB extradiol dioxygenase. Also, the addition of NAD(P) to these reaction
mixtures did not induce decolorization of the meta-cleavage products [50]. To confirm the function of
HcdC, E. coli BL21 cells were transformed with p4pmPmo and pCDF-BC plasmids. The expression of
hcdA, hcdB, and hcdC genes in E. coli cells was confirmed by SDS-PAGE, the enzymes migrated as 62,
3
1, and 20 kDa bands, respectively, as shown in Figure S15 in the Supplementary Material.
The bioconversion of 3-(2,4-dihydroxyphenyl)-propionic acid was conducted in E. coli cells
containing all three recombinant proteins. Later, the reaction mixture was incubated with NH Cl,
4
−
+
and the reaction products were analyzed by HPLC-MS. The ions [M H] and [M+H] with masses
−
of 210.00 and 212.00, respectively, were not detected, compared to the bioconversion mixture with E.
coli cells containing hcdAB genes only. This showed a complete conversion of meta-cleavage product
of the catechol derivative, therefore no picolinic acid derivative could be obtained. No reaction
products of (2E,4E)-2,4-dihydroxy-6-oxonona-2,4-dienedioic acid hydrolysis by the HcdC protein were
identified. We suggest that the later compound was converted to succinic acid (17), which entered
the Krebs cycle, and (E)-2-hydroxy-4-oxopent-2-enoic acid (16), which could be further converted
by E. coli cells, thus complicating the extraction of these reaction products. Nevertheless, it may be
concluded that all three enzymes encoded by the hcdABC operon are responsible for the catabolism of
3
-(2,4-dihydroxyphenyl)-propionic acid in Pseudomonas sp. 7HK4 bacteria.
3
. Discussion
Although coumarins are widely abundant in nature and are intensively used in biotechnology as
precursory compounds [51], the metabolic pathways in microorganisms are still not known in sufficient
detail. In this study, Pseudomonas sp. 7HK4 strain was isolated from soil and it was shown that these
bacteria can utilize only 7-hydroxycoumarin as a sole source of carbon and energy. Several other coumarin
derivatives were also tested, but none of those substrates support the growth of Pseudomonas sp. 7HK4.
However, the experiments with the 7-hydroxycoumarin-induced whole cells show that Pseudomonas sp.
7HK4 has the enzymes that are able to transform coumarin and 6-hydroxycoumarin. The products of these
biotransformations give UV spectra similar to the UV spectrum of 3-phenylpropionic acid, suggesting
that Pseudomonas sp. 7HK4 bacteria can catalyze hydrolysis and reduction of lactone moiety of these
substrates. Furthermore, 3-(2,4-dihydroxyphenyl)-propionic acid has been identified as an intermediate
metabolite during biotransformations of 7-hydroxycoumarin. This finding agrees with the data published
for the conversion of coumarin by Pseudomonas spp., Arthrobacter spp., and Aspergillus spp., which all
catalyze the hydrolysis and reduction of coumarin producing 3-(2-hydroxyphenyl)-propionic acid as
the main intermediate [9–11,14]. However, compared with P. mandelii 7HK described in this study,
little is known about the bioconversion of other coumarin derivatives as well as further conversions of
3-(2-hydroxyphenyl)-propionic acid in bacteria listed above.
The analysis of the 7-hydroxycoumarin-inducible proteins lead to the identification of the genomic
locus hcdABC encoding the enzymes required for 3-(2,4-dihydroxyphenyl)-propionic acid degradation.
A BLAST search uncovered that hcdA, hcdB, and hcdC encode an aromatic flavin-binding hydroxylase,
a ring-cleavage dioxygenase and a hydrolase, respectively. Biochemical analysis of the HcdA
protein confirmed its similarity to class A FAD-binding enzymes (FMOs) [20–23,26–28]. HcdA is
functionally related to previously well-described hydroxylases, such as OhpB monooxygenase from
Rhodococcus sp. V49 [52], MhpA monooxygenase from E. coli K-12 [53] and Comamonas testosteroni
TA441 [54], HppA monooxygenase from Rhodococcus globerulus PWD1 [31], and also para-hydroxybenzoate
hydroxylase (PHBH) from P. fluorescens [55].
HcdA appears to have a high specificity for
-(2,4-dihydroxyphenyl)-propionic acid, converting it to 3-(2,3,5-trihydroxyphenyl)-propionic acid, and a
0-fold lower activity towards trans-2,4-dihydroxycinnamic acid. Other structurally related substrates are
3
4

Molecules 2018, 23, 2613
12 of 19
not used by HcdA. Unlike HcdA, other related FMOs have a broader specificity for substrates. For example,
OhpB monooxygenase is capable to oxidize 2-hydroxy-, 3-hydroxyphenylpropionic and cinnamic acids [52].
The HppA enzyme is more specific to 3-hydroxyphenylpropionic, but 4-chlorophenoxyacetic as well as
4-methyl-2-chlorophenoxyacetic acids are also oxidized [31]. On the other hand, all described FMOs
including HcdA are NAD(P)H dependent, which reduces flavin for the hydroxylation of substrates [20 23].
–
The narrow specificity of HcdA to its natural substrate is typical for class A flavoproteins and shows the
importance of HcdA enzyme in metabolism of 7-hydroxycoumarin. The kinetic analysis of HcdA yields
K
m
values of 50.10
±
3.50
µ
M and 13.00
±
1.20 µM for NADH and 3-(2,4-dihydroxyphenyl)-propionic
acid, respectively. However, no kinetic parameters have been reported for OhpB, MhpA, and HppA
hydroxylases for comparison, though PHBH has been shown to have a kcat of 22.83 s
−1
[56], which is 3-fold
higher than a turnover number for the HcdA enzyme.
The hydroxylated product of the HcdA protein was analyzed by oxidizing it with the HcdB
dioxygenase, followed by a chemical modification to the corresponding derivative of picolinic acid.
1
13
The structure of the later compound was confirmed by H NMR and C NMR spectra. This allowed
the reconstruction of the reaction products of both HcdA and HcdB enzymes. It was shown that
a hydroxylation of 3-(2,4-dihydroxyphenyl)-propionic acid occurs at ipso-position of phenolic ring
followed by internal rearrangements involving (1,2-C,C)-shift (NIH shift) of propionic acid moiety,
hence forming 3-(2,3,5-trihydroxyphenyl)-propionic acid, as shown in Scheme 1. This would explain
both the high specificity of HcdA enzyme for substrates with para-substituted phenol and inability of
Pseudomonas sp. 7HK4 bacteria to utilize coumarin derivatives other than 7-hydroxycoumarin as the
sole source of carbon and energy. Only a few classes of enzymes are able to catalyze ipso-reactions:
laccases, peroxidases, dioxygenases, glutathione S-transferases (GST), cytochrome P450-dependent
monooxygenases (CYP), and flavin-dependent monooxygenases. Among the known examples of
ipso-enzymes, there are dioxygenases from Comamonas testosteroni T2 and Sphingomonas sp. strain
RW1, which are involved in the desulfonation of 4-sulfobenzoate by ipso-substitution [57,58]. A rat
liver CYP system is able to convert p-chloro, p-bromo, p-nitro, p-cyano, p-hydroxymethyl, p-formyl,
and p-acetyl phenols to hydroquinone by ipso-hydroxylation [59]. GST is capable of catalyzing
desulfonylation of sulfonylfuropyridine compounds by nucleophilic attack of the glutathione sulfur
atom at ipso-position [60]. These are the examples of electrophilic or nucleophilic ipso-substitution
reactions, however in some cases, a primary ipso-group is not eliminated, and instead it is shifted
to meta-position. NIH shift restabilizes cyclohexadienone intermediate, because it leads to a
rearomatization [45–48]. We showed that, similarly to flavin-dependent monooxygenases from
Sphingomonas sp. TTNP3 and Sphingobium xenophagum strains, which are responsible for the
degradation of alkylphenols, such as bisphenol A, octylphenol, t-butylphenol, n-octyloxyphenol,
and t-butoxyphenol, HcdA-catalyzed reaction involves a NIH shift. Usually, NIH shift products
are formed during the side reactions, and these internal rearrangements of an alkyl group upon
the ipso-hydroxylation are spontaneous and non-enzymatic in Sphingomonas sp. strains [47,61].
An interesting novelty is that HcdA hydroxylase produces only one product, which has the ipso-group
shifted to the meta-position. Therefore, we propose that in the case of HcdA, NIH shift occurs
enzymatically, but not spontaneously or by a dienone-phenol rearrangement mechanism [62], since all
bioconversions were performed under neutral or basic conditions. Although a further investigation is
needed to determine the exact mechanism of the HcdA enzyme activity.
The analysis of the extradiol dioxygenase HcdB shows that this enzyme has a lower
specificity for substrates. It catalyzes a conversion of the hydroxylated product of the
HcdA enzyme to (2E,4E)-2,4-dihydroxy-6-oxonona-2,4-dienedioic acid, as shown in Scheme 1.
Also, HcdB is capable oxidizing pyrocatechol, 3-methylcatechol, 3-methoxycatechol, 4-methylcatechol,
3
-(2,3-dihydroxyphenyl)-propionic, and caffeic acids using a meta-cleavage mechanism forming yellow
products. HcdB belongs to type I, class II extradiol dioxygenases [24 25], and is functionally related to
OhpD catechol 2,3-dioxygenase from Rhodococcus sp. V49 [52], MhpB extradiol dioxygenase from E. coli
K-12 [53 63], MpcI extradiol dioxygenase from Alcaligenes eutrophus [63], HppB extradiol dioxygenase from
,
,

Molecules 2018, 23, 2613
13 of 19
0
Rhodococcus globerulus PWD1 [31], and DbfB 2,2 ,3-trihydroxybiphenyl dioxygenase from Sphingomonas sp.
RW1 [30].
Finally, we demonstrate that (2E,4E)-2,4-dihydroxy-6-oxonona-2,4-dienedioic acid, the ring
cleavage product of HcdB protein, is subsequently hydrolyzed by the putative HcdC hydroxymuconic
semialdehyde hydrolase. This enzyme has a low sequence homology to any of the previously
characterized enzymes from Rhodococcus sp. V49 [52], E. coli K-12 [53], Comamonas testosteroni
TA441 [55], or Rhodococcus globerulus PWD1 [31], therefore further investigation is needed to elucidate
the exact mechanism of the hydrolysis and the specificity of the HcdC enzyme for substrates.
In summary, here we report a 7-hydroxycoumarin catabolic pathway in Pseudomonas sp. 7HK4 bacteria.
New metabolites and genes responsible for the degradation of 3-(2,4-dihydroxyphenyl)-propionic acid
have been isolated and identified. Our results show that the degradation of 7-hydroxycoumarin in
Pseudomonas sp. 7HK4 involves a distinct metabolic pathway, compared to the previously characterized
coumarin catabolic routes in Pseudomonas, Arthrobacter, and Aspergillus species [9–16]. It has been shown
that Pseudomonas sp. 7HK4 bacteria employ unique flavin-binding ipso-hydroxylase for the oxidation
of the aromatic ring of 3-(2,4-dihydroxyphenyl)-propionic acid. None of the proteins described in
this paper have substantial sequence homology to the previously characterized enzymes implicated
in the degradation of structurally similar substrates, such as 3-(2-hydroxyphenyl)-propionic acid
in Rhodococcus sp. V49 [52], 3-(3-hydroxyphenyl)-propionic acid and 3-hydroxycinnamic acid in
E. coli K-12 [53], Comamonas testosteroni TA441 [54], Rhodococcus globerulus PWD1 [31], or even
4-hydroxyphenylacetate in Escherichia coli W [64]. Thus, our results provide a fundamentally
new insight into the degradation of hydroxycoumarins by the soil microorganisms. In addition,
the discovered new bacteria and enzymes can be further employed for the development of novel
biocatalytic processes useful for industry.
4
. Materials and Methods
4
.1. Bacterial Strains, Plasmids, and Reagents
Pseudomonas sp. 7HK4 bacterial strain capable of using 7-hydroxycoumarin as the sole
source of carbon and energy was isolated from soil by enrichment in mineral medium containing
.05% of 7-hydroxycoumarin. For cloning purposes, E. coli DH5 bacteria ( 80 lacZ M15
(lacZY-argF)U169 deoR recA1 endA1 hsdR17(r -m +) supE44 thi-1 gyrA96 relA1) (Thermo Fischer
0
α
ϕ
∆
∆
K
K
Scientific, Lithuania) were used. E. coli BL21 (DE3) bacteria (F- ompT gal dcm lon hsdS (r -m -)
B
B
B
λ(DE3) [lacI lacUV5-T7 gene 1, ind1, sam7, nin5]) (Novagen, Darmstadt, Germany) were used for
gene expression studies.
All reagents used during this study are listed in Table S1 and plasmids are described in Table S2 in
the Supplementary Material.
4
.2. Bioconversions with Whole Cells
Pseudomonas sp. 7HK4 bacteria were grown in mineral medium supplemented with 0.05% (w/v)
◦
of 7-hydroxycoumarin or glucose, as the sole carbon and energy source, at 30 C with rotary aeration
(
180 rpm) for 48 h. E. coli BL21 (DE3) bacteria containing recombinant genes were grown in 30 mL of
◦
Brain Heart Infusion (BHI) medium at 30 C and 180 rpm overnight. High density bacterial culture was
centrifuged and resuspended in 30 mL of minimal C-750501 medium, in which the synthesis of proteins
was induced with 1 mM of isopropyl-
at 20 C and 180 rpm [65]. Incubation at 20 C was continued for another 24 h. Both Pseudomonas
β
-D-1-thiogalactopyranoside (IPTG) after 1.5 h of incubation
◦
◦
sp. 7HK4 and E. coli cells were sedimented by centrifugation (3220
×
g, 15 min). The collected cells
were washed twice with 15 mL of 0.9% NaCl solution. For whole-cell conversion experiments, cells
from 20 mL of culture were resuspended in 1 mL of 50 mM potassium phosphate buffer (pH 7.2).
All small-scale bioconversions with whole cells were made in 50 mM potassium phosphate buffer, pH
7.2, which contained 1–2 mM of the substrate. The reaction mixtures were kept in a thermoblock at

Molecules 2018, 23, 2613
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◦
3
0 C and 500 rpm. Bioconversion mixtures were centrifuged for 2 min at 10,000
×
g, and 100
µL of the
supernatant were analyzed by UV-VIS spectroscopy (range 200–600 nm). Measurements were repeated
to record the changes in the absorption intensity over time. All measurements were performed with
PowerWave XS microplate reader.
4
.3. Preparation of cell-free extracts
Cells were sedimented by centrifugation (3220
×
g, 15 min). The biomass was resuspended in 3 mL of
5
0 mM potassium phosphate buffer (pH 7.2). The cells were disrupted by pulse-mode sonication (3 min
◦
◦
duration and 1 s cycles) at 4 C. Cell debris was removed by centrifugation (4 C, 16,100× g, 15 min).
4
.4. Protein Purification
Proteins were purified with Äkta purifier 900 chromatography systems (GE Healthcare, Helsinki,
Finland). Cell-free extracts were loaded onto a Ni2+ Chelating HiTrapTM HP column (1–5 mL) (GE
Healthcare, Finland) equilibrated with 50 mM potassium phosphate buffer, pH 7.2, at 1.0 mL/min.
The column was washed with at least 3 volumes of the same buffer. Then the bound proteins
were eluted with 0.5 M imidazole in 50 mM potassium phosphate buffer, pH 7.2. The fractions
containing the purified enzyme were combined and dialyzed against the 50 mM potassium phosphate
◦
buffer, pH 7.2, at 4 C overnight. Proteins were analyzed by SDS-PAGE according to Laemmli [66].
Protein concentration was determined by the Lowry method [67].
4
.5. Preparation of Proteins from a Polyacrylamide Gel for Mass Spectrometric Analysis
Proteins were fractionated on a SDS-polyacrylamide gel. After Coomassie blue R-250 staining,
protein samples were extracted from the gel as described in Reference [68] with minor changes.
Protein bands were excised from the gel with a razor, and the gel was then destained twice with 200
µ
L
◦
of 25 mM ammonium bicarbonate and 50% acetonitrile solution for 30 min at 37 C. Protein disulfide
◦
bonds were reduced with 40
with 30
µ
L of 10 mM dithiothreitol (DTT) for 45 min 60 C, followed by incubation
µ
L of 100 mM iodoacetamide for 1 h at room temperature in the dark to alkylate free cysteines.
Gel slices were washed again twice with 100
solution for 15 min at 37 C, dehydrated by adding 50
centrifuge. Gel pieces were incubated with up to 40
µ
L 25 mM ammonium bicarbonate and 50% acetonitrile
◦
µ
L 100% acetonitrile and dried using a vacuum
◦
µL of activated trypsin (10 ng/µL) at 37 C
overnight. The next day, the supernatant was saved and the peptides were extracted from the gel by
incubating gel slices in two consecutive changes of 50
solution for 1 h at 37 C. Combined supernatants were dried using a vacuum centrifuge at 30 C.
µ
L of 5% trifluoroacetic acid and 50% acetonitrile
◦
◦
Lyophilized peptides were dissolved in 20
µL of 0.1% trifluoroacetic acid solution. Peptides purified
and concentrated using Millipore C18 ZipTips.
4
.6. Enzyme Assay
Activity of 3-(2,4-dihydroxyphenyl)-propionic acid hydroxylase was measured
spectrophotometrically by monitoring absorption changes of the reaction mixture at 340 nm
−1 −1
wavelength due to the oxidation of either NADH or NADPH (ε340 = 6220 M cm ) after the
addition of the substrate. The activity measurements were made with cell-free extracts or the purified
protein. All measurements of the enzyme activity were carried out at room temperature in 1 mL
of reaction mixture, containing 25–50 mM tricine or potassium phosphate buffers (pH 7.8), 100
NADH or NADPH, and 150 M aromatic substrate. One unit of activity was defined as the amount of
the enzyme that catalyzed the oxidation of 1 µmol of NADH or NADPH per minute.
µM
µ
4
.7. In vivo Bioconversion of 3-(2,4-Dihydroxyphenyl)-propionic Acid
E. coli BL21 (DE3) bacteria, containing p4pmPmo and pTHPPDO plasmids, were grown in 200 mL
◦
of BHI medium at 30 C and 180 rpm overnight. High density bacterial culture was centrifuged and

Molecules 2018, 23, 2613
15 of 19
resuspended in 200 mL of minimal C-750501 medium, in which synthesis of proteins was induced with
◦
1
mM of IPTG at 20 C and 180 rpm [65]. After 24 h of induction 3-(2,4-dihydroxyphenyl)-propionic
acid was added to the final concentration of 4 mM. Bioconversion mixture was incubated for another
◦
3
days at 30 C with shaking. Cells were removed by centrifugation for 30 min at 3220
×
g and the
◦
supernatants were kept at 4 C.
4
.8. Purification of 3-(2,4-Dihydroxyphenyl)-propionic Acid Bioconversion Product
The supernatant containing the 3-(2,4-dihydroxyphenyl)-propionic acid oxidation product was
incubated with 1.2 M ammonium chloride at room temperature overnight [39 50]. Reaction mixture
,
was concentrated to ~100 mL volume and adjusted to pH 1 with concentrated HCl. The remains of
substrate were extracted by five consecutive changes of 25 mL of ethyl acetate, and then aqueous
fraction was purified using a reverse phase C18 column, equilibrated with water. Column was washed
with at least 100 mL of water and then eluted with linear gradient of 0–60% methanol solution at a flow
◦
rate of 2 mL/min. Aqueous fractions were combined and evaporated (40 C). Brownish crystals were
dissolved in 0.1% formic acid solution and again loaded onto a reverse phase C18 column, previously
equilibrated with 0.1% formic acid solution. Column was washed with at least 30 ml of 0.1% formic
acid solution and then eluted with 60% methanol solution. Picolinic acid derivative was eluted with
0
.1% formic acid solution. Fractions containing the product were collected, combined, and evaporated
◦
(40 C). Picolinic acid yield from 145 mg of 3-(2,4-dihydroxyphenyl)-propionic acid fermentation was
3
4 mg, 24% of the theoretical yield. The product had traces of formic acid impurities, which aided the
dissolution of the analyte in D O for NMR analysis.
2
4
.9. High-Performance Liquid Chromatography and Mass Spectrometry
Before the analysis, the samples were mixed with an equal part of acetonitrile and centrifuged.
High-performance liquid chromatography and mass spectrometry (HPLC-MS) was carried out using
the system, consisting of the CBM-20 control unit, two LC-2020AD pumps, SIL-30AC auto sampler,
and CTO-20AC column thermostat, using the SPD-M20A detector and LCMS-2020 mass spectrometer
with ESI source (Shimadzu, Kyoto, Japan).
Chromatographic fractionation was conducted using YMC-Pack Pro C column, 150
×
3 mm
YMC, Kyoto, Japan) at 40 C, with 0.1% formic acid solution in water and acetonitrile gradient from
% to 95%.
Mass spectra were recorded from m/z 10 up to 500 m/z at 350 C and
18
◦
(
5
◦
±
4500 V using N2.
Mass spectrometry analysis was carried out using both the positive and negative ionization modes.
The data were analyzed using LabSolutions LC/MS software (Shimadzu, Japan).
4
.10. Nuclear Magnetic Resonance Spectroscopy
In total, 20 mg of the sample was dissolved in 0.5 mL D O. NMR spectra were recorded on an
2
1
13
Ascend 400: H NMR – 400 MHz, C NMR – 100 MHz (Bruker, Billerica, MA, USA). Chemical shifts
are reported in parts per million relative to the solvent resonance signal as an internal standard.
4
.11. Protein MS-MS Analysis
Peptides were subjected to de novo sequencing based on matrix-assisted laser desorption
ionization time of flight (MALDI-TOF/TOF) mass spectrometry (MS) and subsequent computational
analysis at the Proteomics Centre of the Institute of Biochemistry, Vilnius University (Vilnius, Lithuania).
The sample was purified as described previously in Materials and Methods. Tryptic digests (0.5
were transferred on 384-well MALDI plate with 0.5 L 4 mg/mL -cyano-4-hydroxycinnamic acid
CHCA) matrix in 50% acetonitrile with 0.1% trifluoroacetic acid and analyzed with an Applied
Biosystems/MDS SCIEX 4800 MALDI TOF/TOF mass spectrometer. Spectra were acquired in the
positive reflector mode between 800 and 4000 m/z with fixed laser intensity at 3700 (Laser shots: 400;
Mass accuracy: 50 ppm). The most intense peaks of each survey scan (MS) were fragmented for
µL)
µ
α
(
™
±

Molecules 2018, 23, 2613
16 of 19
sequence analysis (Collision energy: 1 keV; CID: no CID or medium air pressure CID used; Laser
intensity: 4200–4400; Laser shots: 500–1000; Fragment mass accuracy:
and peak lists were generated using GPS Explorer™ De Novo Explorer.
±
0.1 Da). Sequence analysis
4
.12. DNA Sequencing and Accession Numbers
The genome sequencing by Illumina (paired-ends) and assembling (in total, 35 contigs) was
carried at Baseclear (Leiden, The Netherlands). The FASTQ sequence reads were generated using
the Illumina Casava pipeline version 1.8.3. Initial quality assessment was based on data passing
the Illumina Chastity filtering. Subsequently, reads containing adapters and/or PhiX control signal
were removed using an in-house filtering protocol. The second quality assessment was based on
the remaining reads using the FASTQC quality control tool version 0.10.0. The number of reads was
5
,605,132 and an average quality score (Phred) was 37.72.
The accession number for partial 16S ribosomal RNA nucleotide sequence of Pseudomonas sp.
HK4 is MH346031. Accession numbers for the sequences of hcdA, hcdB, and hcdC genes are MH346032,
7
MH346033, MH346034, respectively.
Supplementary Materials: The following are available online, Table S1: Materials and reagents used in the
studies, Table S2: Plasmids used in the studies, Table S3: The list of primers used in this study, Figure S1:
Phylogenetic tree of Pseudomonas sp. 7HK4 bacteria based on partial 16S rDNA sequences, Figure S2:
Biotransformation of 7-hydroxycoumarin, 6-hydroxycoumarin, coumarin and 6,7-dihydroxycoumarin by whole
cells of Pseudomonas sp. 7HK4, Figure S3: Phylogenetic trees of HcdA, HcdB and HcdC proteins, Figure S4:
SDS-PAGE, UV/Vis spectrum and analytical gel filtration chromatography of purified HcdA protein, Figure S5:
Specificity of HcdA protein to flavin and nicotinamide cofactors, Figure S6: Activity of HcdA protein in
different buffer systems, Figure S7: Activity of HcdA protein in different pH, Figure S8: Kinetic analysis
of HcdA as determined by NADH oxidation for 3-(2,4-dihydroxyphenyl)-propionic acid, Figure S9: Kinetic
analysis of HcdA as determined by NADH oxidation for NADH, Figure S10: Double reciprocal plot of NADH
oxidation as a function of NADH concentration, Figure S11: Biotransformations of 3,4-dihydroxybenzoic acid,
0
0
0
2
,3 -dihydroxy-4 -methoxyaceto-phenone hydrate, gallacetophenone, pyrogallol, 2,3,4-trihydroxybenzoic acid
1
and 2,3,4-trihydroxy-benzophenone by whole cells of E. coli BL21 containing hcdB gene, Figure S12: H NMR
1
3
spectrum of 6-(2-carboxyethyl)-4-oxo-1,4-dihydropyridine-2-carboxylic acid, Figure S13: C NMR spectrum of
6
-(2-carboxyethyl)-4-oxo-1,4-dihydropyridine-2-carboxylic acid, Figure S14: Resonance structure of oxo-picolinic
acid derivative showing electron densities on aromatic carbons, Figure S15: SDS-PAGE of E. coli BL21 cell-free
extract, containing induced recombinant HcdA, HcdB and HcdC proteins, Figure S16: HPLC-MS analysis of
3
-(2-hydroxyphenyl)-propionic acid bioconversion mixture.
Author Contributions: Conceptualization and Funding Acquisition, R.M.; Investigation, A.K.; Data Analysis,
A.K. and R.M.; Writing, Review & Editing, A.K. and R.M.
Funding: This research was funded by the European Union’s Horizon 2020 research and innovation program
[
BlueGrowth: Unlocking the potential of Seas and Oceans] under grant agreement No. 634486 (project
acronym INMARE).
Acknowledgments: We are grateful to Marija Ger for performing peptide sequence analysis, Daiva Tauraite for
˙
help with chemical synthesis, and Laura Kalinien e˙ for critical reading of the manuscript.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the plasmid DNAs are available from the authors.
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