Engineered Bacterial Flavin‐Dependent Monooxygenases for the Regiospecific Hydroxylation of Polycyclic Phenols

Abstract 4‐Hydroxyphenylacetate 3‐hydroxylase (4HPA3H), a flavin‐dependent monooxygenase from E. coli that catalyzes the hydroxylation of monophenols to catechols, was modified by rational redesign to convert also more bulky substrates, especially phenolic natural products like phenylpropanoids, flavones or coumarins. Selected amino acid positions in the binding pocket of 4HPA3H were exchanged with residues from the homologous protein from Pseudomonas aeruginosa, yielding variants with improved conversion of spacious substrates such as the flavonoid naringenin or the alkaloid mimetic 2‐hydroxycarbazole. Reactions were followed by an adapted Fe(III)‐catechol chromogenic assay selective for the products. Especially substitution of the residue Y301 facilitated modulation of substrate specificity: introduction of nonaromatic but hydrophobic (iso)leucine resulted in the preference of the substrate ferulic acid (having a guaiacyl (guajacyl) moiety, part of the vanilloid motif) over unsubstituted monophenols. The in vivo (whole‐cell biocatalysts) and in vitro (three‐enzyme cascade) transformations of substrates by 4HPA3H and its optimized variants was strictly regiospecific and proceeded without generation of byproducts.

Activity assay for PrnF 5 Spectrophotometric activity assay for 4HPA3H 5 Determination of shunt H2O2 produced by uncoupled NADH consumption 5 In vitro conversion of monophenols 6 References 6 Supplementary figures and tables 7 Figure S1. SDS-PAGE of cells expressing 4HPA3H and variants 7 Figure S2. SDS-PAGE of purified enzymes used in in vitro hydroxylation 7 Figure S3. Catechol complex formation assay for determination of 4HPA3H activity 8 Figure S4. Dependence of the in vitro conversion of p-coumaric acid on the FAD and NADH concentration 8 Table S1. In vivo conversion data (corresponding to Fig. 2) 9 Table S2. Amount of hydrogen peroxide produced during hydroxylation of p-coumaric acid 1 under in vitro conditions 11  (14) 19 4-(3,4-Dihydroxyphenyl)-butan-2-one (16) 20 estimated using molecular markers from Fermentas (Waltham, USA). The enzymes were stored at -80 °C until use.

Protein determination
The protein concentration was determined by the method of Bradford. [5] Protein assays in microplate scale were carried out with commercial Bradford reagent (Roth, Karlsruhe, Germany) according to manufacturer´s instructions. Protein concentrations were calculated using standard curves of bovine serum albumin.

Whole-cell bioconversions
Reactions were performed in 50 ml Erlenmeyer flasks. 10 ml of auto-inducing medium [2] containing hydroxylase substrate (200 µM, added from stock solutions (200 mM) in methanol) and kanamycin (50 µg ml -1 ) were inoculated with E. coli BL21(DE3) harboring either the plasmids for production of 4HPA3H or its variants (hydroxylation reactions) or the empty vector (controls). Cells were grown at 37 °C with shaking (200 rpm). After 16 hours of incubation, samples (1.5 ml) were taken. For all conversions except those of 2-hydroxycarbazole, the samples were centrifuged at 14,000 g for 10 min. The supernatants (1 ml) were acidified by the addition of 10 µl of formic acid and extracted two times with 500 µl of ethyl acetate. For samples containing the poorly soluble 2-hydroxycarbazole, the centrifugation step was omitted, and the cell suspension was directly used in the extraction procedure. After extraction the organic phases were combined, the solvent was removed by vacuum centrifugation and the residue was re-dissolved in acetonitrile (200 µl). This solution was used for HPLC measurements (see Section below). The data shown in this study were generated from two independent experiments.
For product identification, preparative transformations were performed similar to the reactions described above but in larger scale, i.e. in 2,000 ml Erlenmeyer flasks containing 500 ml of medium. After incubation at 37 °C for 16 hours, formic acid (5 ml) was added to the cell suspension (conversion of 2-hydroxycarbazole) or to the supernatant from centrifugation (8,000 g, 10 min) (all other conversions). The combined organic phases from extraction with ethyl acetate (2 x 250 ml) were dried with anhydrous Na2SO4, filtered and the solvent was removed under reduced pressure. The residue was re-dissolved in a minimum volume of methanol and used for preparative HPLC (see below).

HPLC
Samples (10 µl) from whole-cell biotransformation (see above) or from in vitro assays (see below) were applied to a C18 reversed-phase column (Pack ODS-A, 5 µm, 150 x 4.6 mm, YMC Europe Dinslaken, Germany) on a Merck Hitachi HPLC system. The compounds were separated by elution with acetonitrile-water (containing 0.2 % (v/v) formic acid). The following gradient was used at a flow of 0.8 ml min -1 (percentage of acetonitrile in v/v): 5 % for 4 min, 5 to 100 % for 21 min, 100 % for 5 min, 100 to 5 % for 1 min and 5 % for 5 min.
All products that were generated in large-scale biotransformations (see above) were separated on a preparative HPLC system by using a C18 reversed-phase column (Column ODS-A, 5 µm, 150 x 20 mm, YMC Europe, Dinslaken, Germany). The following gradient was used at a flow of 15 ml min -1 (percentage of acetonitrile in v/v): 50 % for 4 min, 50 to 100 % for 21 min, 100 % for 5 min, 100 to 50 % for 1 min and 50 % for 5 min.
For both analytical and preparative HPLC, elution was monitored at a wavelength of 280 nm.
Reaction products were identified by means of authentic standards, which were applied in different concentrations for quantification, or in case of 4-(3,4-dihydroxyphenyl)-butan-2-one (16) and 2,3-dihydroxycarbazole (12) by NMR and ESI-MS analysis of reaction products obtained from preparative whole-cell transformations (as described above).

NMR and ESI-FTMS analysis
The 1 H NMR spectra were recorded on a 400 MHz Agilent DD2 400 NMR spectrometer at 25 °C. Substances were dissolved in CD3OD, and chemical shifts were referenced to internal tetramethylsilane. The data (see page 10) were evaluated by Mestrelab Research S.L.
The negative ion high resolution ESI mass spectra (see page 10) and the collision induced dissociation (CID) MS 2 measurements were obtained from a Orbitrap Elite mass spectrometer

Activity assay for PrnF
The activity of the flavin reductase component PrnF was measured by the decrease of the absorbance at 339 nm (ε340 = 6.22 mm -1 cm -1 ) [7] due to the oxidation of NADH. The reaction was performed in a cuvette with a light path of 1 cm that contained reaction buffer (100 mM Tris/HCl, pH 7.5), NADH (200 µM) and FAD (30 µM) in a total volume of 1 ml. The reactions were started by the addition of 12.5 µg ml -1 PrnF to the reaction mixture. An assay mixture without enzyme was used as a blank. The decrease in absorbance was recorded continuously at intervals of 40 s for a total reaction time of 10 min. One unit of enzyme activity represents the amount of enzyme converting 1 µmol of NADH in one minute of reaction time.
The data shown in this study are the means of three independent measurements.
Reactions were started by addition of PrnF (566 mU ml -1 ) and hydroxylase (1 mg ml -1 ). The reactions were stopped after distinct times of incubation (0 -240 min) at 25 °C by mixing with 40 µl of catechol reagent (2 mM FeCl3 in 10 mM HCl, 1 mM Triton X-100) to generate a colored Fe 3+ /product complex. From the absorbance at 595 nm, measured after 5 min of equilibration at room temperature, the concentration of formed product was calculated by means of a standard curve taken with mixtures of the substrate (x mM p-coumaric acid or ferulic acid) and the corresponding product (2-x mM caffeic acid or 5-hydroxy-ferulic acid). One unit of enzyme activity represents the amount of hydroxylase converting 1 µmol of p-coumaric acid in one minute of reaction time. The data shown in this study are the means of two independent experiments.

Determination of shunt H2O2 produced by uncoupled NADH consumption
Microplate reactions (80 µl) were performed as described in the Section "Spectrophotometric activity assay for 4HPA3H", except that the substrate p-coumaric acid was added from an aqueous stock solution of its sodium salt and that the reactions contained less enzyme (0.25 mg ml -1 wildtype enzyme or 4HPA3H Y301F/S462A) and an optimized amount of FAD (10 µM) and NADH (200 µM). Twelve independent reactions were set up per enzyme. After 60 min of incubation at 25 °C, four of the replicates were treated with catechol reagent (see Section above) to determine the formed caffeic acid. Four other replicates were used for determination of H2O2 using an adaptation of the method described by Dippe and Ulbrich-Hofmann. [8] Therefore, the reactions were mixed with 20 µl of a solution containing chromogenic peroxidase substrates (5 mM 4-aminoantipyrine, 10 mM phenol, 100 mM Tris/HCl, pH 7.5) and 6 20 µl of horseradish peroxidase (50 µg ml -1 in 100 mM Tris/HCl, pH 7.5). The absorbance at 490 nm caused by the formation of the quinoneimine dye was measure after 10 min of incubation at 25 °C. For the calculation of the H2O2 concentration the difference in absorbance to the remaining four independent reactions, which were treated with water instead of peroxidase, was used. The calculation was based on a standard curve (0 -500 µM H2O2 in 100 mM Tris/HCl, pH 7.5) taken under similar conditions.

In vitro conversion of monophenols
The reactions (200 µl) were performed under optimized conditions in 1.5 ml reaction tubes.