*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, 2-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 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* (**h**ydroxy**c**oumarin **d**egrading 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].

## *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 His6-tagged protein was produced in *Escherichia coli* BL21, and purified 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. The highest rate of oxidation of NADH was recorded in the presence of 3-(2,4-dihydroxyphenyl)-propionic acid. A 40-fold lower rate was observed when *trans*-2,4-dihydroxycinnamic acid was used as the substrate. The HcdA was not active towards *trans*-cinnamic, *cis*-2,4-dihydroxycinnamic, 3-(2-hydroxyphenyl)-propionic, 3-(2-hydroxyphenyl)-2-propenoic, 3-(4-hydroxyphenyl)-2-propenoic, 3-(3-hydroxyphenyl)-2-propenoic, 3-(2-bromophenyl)-propionic, 3-(2-nitrophenyl)-propionic, and 3-phenylpropionic acids, cinnamyl alcohol, 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 His6-tag did not affect the enzymatic activity of 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 340 and 490 nm upon addition of 3-(2,4-dihydroxyphenyl)-propionic acid to the reaction mixture. 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]<sup>−</sup> 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.

**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]<sup>−</sup> generated are at *m*/*z* 181 (3-(2,4-dihydroxyphenyl)-propionic acid), 195 (product of 3-(trihydroxyphenyl)-propionic acid autooxidation), and 363 (dimer of 3-(2,4-dihydroxyphenyl)-propionic acid).
