Expanding the Substrate Scope of Nitrating Cytochrome P450 TxtE by Active Site Engineering of a Reductase Fusion

Abstract Aromatic nitration reactions are a cornerstone of organic chemistry, but are challenging to scale due to corrosive reagents and elevated temperatures. The cytochrome P450 TxtE nitrates the indole 4‐position of l‐tryptophan at room temperature using NO, O2 and NADPH, and has potential to be developed into a useful aromatic nitration biocatalyst. However, its narrow substrate scope (requiring both the α‐amino acid and indole functionalities) have hindered this. Screening of an R59 mutant library of a TxtE‐reductase fusion protein identified a variant (R59C) that nitrates tryptamine, which is not accepted by native TxtE. This variant exhibits a broader substrate scope than the wild type enzyme and is able to nitrate a range of tryptamine analogues, with significant alterations to the aromatic and aminoethyl moieties.


Chemicals and reagents
All chemicals and reagents were purchased from Sigma-Aldrich, Fisher Scientific, Alfa Aesar, VWR International, Acros Organics or Fluorochem and were used as supplied unless stated otherwise.
Solvents were sourced from either Sigma-Aldrich or Fisher Scientific. Thin-layer chromatography was performed on silica-coated aluminium from Merck (TLC silica gel 60 F254) and visualised using UV fluorescence and/or ninhydrin stain. Silica column chromatography was performed using silicon S2 dioxide from Sigma-Aldrich (40-63 µm, 60 Å pore size). NMR solvents were purchased from Sigma-Aldrich or Cambridge Isotope Laboratories.

Instrumentation
NMR spectra were obtained on either a Bruker Avance III 400 MHz spectrometer at 400 MHz ( 1 H) and 100 MHz ( 13 C), or a Bruker Avance III 500 MHz spectrometer equipped with a DCH cryoprobe for at 500 MHz ( 1 H) and 125 MHz ( 13 C). Spectra were recorded at 25 ̊ C in D2O, DCl in D2O or MeOD.
Chemical shifts are reported as δ values in ppm relative to the residual HOD (4.80 ppm ( 1 H)) or CH3OD (3.34   DNA and protein concentrations were measured using a Thermo Scientific NanoDrop Lite spectrometer. Cell growth and protein expression was conducted in New Brunswick Scientific Innova44 S3 incubators and cells were lysed using a Constant Systems cell disruptor. Cell optical density was measured using a Thermo BioMate3 spectrometer at 600 nm.

Plasmids, bacterial strains and enzymes
The construction of pSB13, containing the gene for txtE from Streptomyces turgidiscabies Car8 in pET-151, has been described previously. 1 The gene encoding TxtE-BM3R, codon-optimised for E. coli, cloned as a His6 translational fusion into pET28a(+) was purchased from GenScript, NJ, USA.
Competent cells were were either Invitrogen One Shot TOP10 and BL21 Star (DE3) or New England Biolabs DH5α and BL21(DE3). Restriction enzymes were purchased from either ThermoFisher Scientific or New England Biolabs.

Biological reagents and media
Miller's Luria-Bertani (LB) media was purchased from Fisher Scientific and prepared to 25 g/L in deionised water. Solid LB-agar media was prepared using 25 g/L LB broth and 15 g/L bacto agar in deionised water. Terrific broth (TB) was purchased from VWR and prepared by adding 50 g of powder to 990 mL deionised water and 10 mL glycerol prior to autoclaving. Ampicillin and kanamycin stock solutions were prepared to 100 mg/mL and 50 mg/L, respectively, and filter sterilised prior to being stored at -20 ̊ C until use. Isopropyl β-D-1-thiogalactopyranoside (IPTG) purchased from VWR was prepared as a 0.5 M stock solution in deionised water, filter sterilised and stored at -20 ̊ C until use.

General procedure for protein expression
The general procedure for the expression of His6-TxtE-BM3R and His6-TxtE-BMR3(R59C) is as follows: the construct was used to transform E. coli BL21Star (DE3). A single transformant from antibiotic-supplemented LB-agar plates was used to inoculate LB medium (10 mL) supplemented with kanamycin (50 µg/mL) and the resulting culture was incubated at 30 ºC overnight with shaking at 220 rpm. This overnight culture (5-10 mL) was used to inoculate LB or TB medium (1 L) supplemented with antibiotic and was incubated at 37 ºC with shaking at 220 rpm until the optical density at 600 nm reached 0.6-0.8. The culture was allowed to cool and isopropyl-β-D-thiogalactopyranoside (IPTG) (0.1 mM), 5-aminolevulinic acid hydrochloride (5-ALA) (1 mM), iron (III) chloride (5 mg/L) and thiamine (1 mM) were added. The culture was incubated overnight at 15 ºC with shaking at 220 rpm. Cells from the overnight incubation were harvested by centrifugation (4000 x g, 30 min), re-suspended in 10 mL Buffer A (table S1) and phenylmethylsulphonyl fluoride (PMSF, 1 mM final conc.) was added. The cells were lysed and cell debris was pelleted by centrifugation (11,000 x g, 30 min). The supernatant was passed through a 0.2 µm syringe filter and applied to a 1 mL HiTrap™ HP affinity column (Nickel S4 Sepharose High Performance, GE Healthcare) equilibrated with Buffer A. Unbound proteins were eluted with Buffer A (10 mL) and His6-TxtE-BM3R and the R59C mutant were eluted using 5, 3, 3, and 3 mL of buffers B1-B4, respectively. The orange fractions were pooled and concentrated to ~ 300 µL (Ultra centrifugal device 50 kDa membrane) before being diluted with Buffer C and concentrated again. This concentration procedure was repeated twice more before the purity of the collected fractions was analysed by SDS-PAGE (10%), and the purified protein was flash frozen in aliquots using liquid nitrogen for storage at -80 ºC until further use.

Protein analysis and characterisation
SDS-PAGE analysis of purified proteins was conducted using a 10% acrylamide/bis-acrylamide gel.
The loading gel was allowed to set for 30 minutes prior to the addition of the stacking gel plus comb.
This was set for at least 30 minutes before conducting the analysis. The electrophoresis tank was filled with running buffer (2 M glycine, 1% SDS, 250 mM Tris-HCl, pH 8.0) and the set gel was appropriately placed inside. Protein samples (10-20 µL) were prepared firstly by the addition of loading dye (2-4 µL, 5X stock contains 250 mM Tris·HCl, pH 6.8, 10% SDS, 30% (v/v) Glycerol, 10 mM DTT, 0.05% (w/v) Bromophenol Blue). The samples were heated to 80 ºC for 5 minutes before being applied to the SDS-PAGE gel lane alongside protein ladder (3 μL, Thermo Scientific Page Ruler Plus Prestained). The gel was run at 200 V for 35 minutes and visualised using InstantBlue Protein Stain (Expedeon).
An estimate of the percentage of active heme-containing protein in the solution was calculated by UVvisible spectroscopy. A solution of the purified protein (10-20 µL) was added to a cuvette containing Tris-HCl (25 mM, pH 8.0) and a spectrum was measured between 200 and 500 nm using a blank cuvette containing Tris-HCl (25 mM, pH 8.0) as a reference. The ratio (A420nm/A280nm) x 100, was used as an estimate for the percentage of total incorporated heme and was used to calculate an accurate yield of active cytochrome P450 in the protein solution.
Total protein concentration was calculated using a Nanodrop Lite instrument with the absorbance units set to 1 mg/mL. Using the Beer-Lambert law, A = εlc, where A is the measured absorbance, l is the path length (1 cm), and ε is the predicted extinction coefficients. The protein concentration, c, was calculated for each protein prior to use.

Generation of TxtE-BM3R mutant library
The codon for R59 in the His6-TxtE-BM3R construct was randomised in one step using the Q5-site directed mutagenesis protocol (New England Biolabs). The three mutagenic forward primers encoding NDT, VHG and TGG at the site of saturation were mixed in a ratio of 12:9:1, respectively, to a final concentration of 10 µM. The PCR reaction mixture (50 µL final volume) contained 25 µL Q5 hot start polymerase, 2.5 µL forward primer mixture (10 µM, GACCGCGGACNDTGGTACCGAGG, GACCGCGGACVHGGGTACCGAGG, GACCGCGGACTGGGGTACCGAGG), 2.5 µL reverse S5 primer (10 µM, AGACGCGGGTCCTTCAGACCCG), 2 µL template DNA (10 ng/µL) and 18 µL nuclease-free water. The PCR reaction mixture (50 µL) was split into 4 x 12.5 µL reactions accounting for the different annealing temperatures required (72.0, 70.2, 65.4 and 61.0 ºC). The PCR program consisted of an initial denaturation step at 98 ºC for 30 s, followed by 25 cycles of 98 ºC for 10 s, annealing at the given temperatures for 30 s, elongation at 72 ºC for 4 min 30 s, followed by a final elongation step at 72 ºC for 2 min. The PCR reaction mixtures were combined and 1 µL was added to 5 µL of KLD reaction buffer, 1 µL of 10X KLD enzyme mix and 3 µL nuclease-free water. The mixture was incubated for 5 min at room temperature before being used to transform 5-alpha competent E. coli (New England Biolabs). The next day the transformants were rinsed from the agar plate (50 µg/mL kanamycin) using 1 mL LB broth and plasmid DNA was extracted using a DNA miniprep kit (Qiagen).
Randomisation at the site of saturation was confirmed for each library by DNA sequencing.

High-throughput screening and analysis of the His6-TxtE-BM3R mutant libraries
The site-randomised DNA mixture was used to transform E. coli BL21 (DE3) competent cells shaken at 800 rpm and 20 ºC for 10 min before being centrifuged (4000 x g). 200 µL of the supernatant was transferred to a 96-shallow well plate for analysis by LC-MS (elution profile is given in Table S2).

In vitro activity assays of His6-TxtE-BMR3(R59C) with tryptamine and analogues
The ability of TxtE-BM3R_R59C to nitrate several tryptamine analogues was tested by preparing the following reactions in 2 mL Eppendorf tubes: 10 µM enzyme in 25 mM Tris-HCl, pH 8.0, containing 2.0 mM substrate 1-14 or L-Trp, 2.4 mM NADPH and 4.0 mM DEANO in a final volume of 100 µL.
The reaction was incubated at 20 ºC for 3 h, then quenched using 100 µL methanol. The mixture was filtered using 0.2 µM microspin filter tubes (Corning) and analysed by LC-MS using the elution profile given in Table S3. Following initial LC-MS analysis the molecular formulae of nitrated 1-5, 10 and Ltryptophan were confirmed by UHPLC-ESI-Q-ToF MS analysis.
A single transformant harbouring plasmid, pET28a-(+)-txtE-bm3r, from antibiotic-supplemented LBagar plates was used to inoculate LB medium (20 mL) supplemented with kanamycin (50 µg/mL). The culture was incubated at 30 ºC overnight with shaking at 220 rpm. 5 mL of this overnight culture was used to inoculate 4 x 1 L of TB medium supplemented with antibiotic and the resulting culture was incubated at 37 ºC with shaking at 220 rpm until the optical density at 600 nm reached 0.6-0.8. The cultures were allowed to cool and isopropyl-β-D-thiogalactopyranoside (IPTG) (0.1 mM), 5aminolevulinic acid hydrochloride (5-ALA) (1 mM), iron (III) chloride (5 mg/L) and thiamine (1 mM) were added. The cultures were incubated overnight at 18 ºC with shaking at 220 rpm and the cells were harvested by centrifugation (4000 x g, 30 min). The pellets were re-suspended in 30 mL Buffer A (Table   S1) and phenylmethylsulphonyl fluoride (PMSF, 1 mM final conc.) was added. The cells were lysed and cell debris was pelleted by centrifugation (11,000 x g, 30 min). The supernatant was passed through a 0.2 µm syringe filter and applied to a 5 mL HiTrap™ HP affinity column (Nickel Sepharose High Performance, GE Healthcare) equilibrated with Buffer A. Unbound proteins were eluted with Buffer A (30 mL) and His6-TxtE-BM3R(R59C) was eluted using 15, 10, 10, and 10 mL of buffers B1-B4, respectively. The orange fractions were pooled and concentrated to 1 mL (Ultra centrifugal device 50 kDa membrane) before being diluted with 20 mL Reaction Buffer (25 mM Tris-HCl, pH 8.0 and 100 mM NaCl) and concentrated again. Concentration was repeated twice more to ensure complete removal of imidazole from the protein solution. The His6-TxtE-BM3R(R59C) protein solution (1 mL, 48 S7 mg/mL) was diluted with a further 12 mL Reaction Buffer (RB) in a 50 mL falcon tube, and tryptamine (0.6 mL, 50 mM in 10 % DMSO in RB) and NADPH (0.72 mL, 50 mM in RB) were added. The mixture was stirred at 20 ºC for 5 min, DEANO (0.6 mL, 0.1 M in 10 mM NaOH) was added to start the reaction and the tube was sealed. After 20 h the reaction was quenched with 10 % HCl in MeOH (15 mL), stirred for 30 min and filtered to remove precipitated proteins. The filtrate was concentrated in vacuo and freeze-dried to remove the remaining liquid. The residue was dissolved in 2 mL H2O and purified by HPLC (Table S4).

Qualitative binding assaydetermining protein-substrate binding type
The protein was added to a cuvette containing Tris-HCl (25 mM, pH 8.0) to a concentration of 8 µM.
A reference spectrum was recorded between 300 and 500 nm to provide a reference. L-tryptopan or tryptamine were to a final concentration of 2 mM, the spectrum was re-recorded and the reference spectrum was subtracted to give the difference spectrum.

Determination of substrate dissociation constants
An 8 µM solution of the protein was prepared in Tris-HCl (25 mM, pH 8.0). A solution of the substrate (0.5-1.0 µL titrations, 10-50 mM stock) was added stepwise to the cuvette (final concentration 0-10 mM) and a spectrum was recorded after each addition. A corresponding reference spectrum for each measurement was recorded and subtracted from the measured spectrum. This procedure was repeated until the total volume of substrate solution added to the cuvette was no greater than 5% of the volume of the protein solution. The experiment was conducted in least duplicate for each substrate tested.
The absorbance at λmin was subtracted from the absorbance at λmax and plotted against the concentration of substrate. The data were fitted to the Michaelis-Menten equation using OriginPro 9.1, and the dissociation constants were calculated from the resulting binding curves.  12 (1H, s). These data match those reported in the literature. Note that the signal due to the α-proton is obscured by a large water peak resulting from HCl added to the solution to improve the solubility of the sample.

Synthesis of 4-nitrotyptamine [3]
To a stirred solution of 2-cyclohexan-1-one in cyclohexanol (1 % v/v 0.6 mL), 4-nitro-L-tryptophan (41 mg, 0.16 mmol) was added and the resulting suspension was heated to 154 °C under reflux. After 2 hours the reaction was cooled to room temperature and diluted with chloroform (1 mL). The solution was passed through a silica gel pad and washed with water (2 x 2 mL). The organic layer was concentrated in vacuo and further diluted with methanol (1 mL) before being purified by reversed phase HPLC (see Table S4) to give 4-nitrotryptamine (7 mg, 0.034 mmol, 21 %). 1
After 2 h, the reaction was cooled to room temperature and diluted with chloroform (1 mL). The dark brown solution was passed through a silica gel pad and then washed with water (2 x 2 mL). The organic layer was concentrated in vacuo to give the desired product as a pale brown solid (22.6 mg, 0.13 mmol, 32 %).   NdeI  CATATGGTGACCGTGCCGAGCCCGCTGGCGGACCCGAGCATTGTGCCGGACCCGTACCCGGTTTACGCGGACCTGGCGC  AACGTCGCCCGGTTCACTGGGTTGAACGTCTGAACGCGTGGGCGGTGCTGACCTACGCGGATTGCGCGGCGGGTCTGAA  GGACCCGCGTCTGACCGCGGACCGTGGTACCGAGGTTCTGGCGGCGAAATTCCCGGGTCAGCCGCTGCCGCCGGACAAC  ATCTTTCACCGTTGGACCAAGAACGTGGTTATGTATACCGATCCGCCGCTGCATGATGCGCTGCGTCGTAGCGTGCGTGC  GGGTTTCACCCGTGCGGCGCACCAACACTATGATCAGGTGCTGCAAAAAGTTGCGCATGACCTGGTTGCGAGCATCCCGG  CGGGTGCGACCGAAATTGATGCGGTGCCGGCGCTGGCGGCGGAGCTGCCGGTGCGTAGCGCGGTTCATGCGTTCGGTG  TTCCGGAGGAAGACCTGGGCTTTCTGATCCCGCGTGTGAACACCATTATGACCTACCACAGCGGTCCGAAGGATCAGCCG  GTTACCCAAGAAATCATTCTGGAGAAACTGACCGACCTGCACACCTATGCGAGCGAGCTGCTGCAGGGCATGCGTGGCA  AGGTGCTGCCGGATACCGTTATTGCGCGTCTGGCGGCGGCGCAAGATGGTCTGACCGAAACCACCCCGGAGCAGACCGT  TCACCAACTGGCGCTGGTGTTCATTGCGCTGTTTGCGCCGACCACCCCGGGTAGCCTGAGCAGCGGTACCCTGGCGTTTG  CGCGTAACCCGCGTCAGGTGGAACGTTTTCTGGCGGATCAAGCGTGCGTTGACAACACCGCGAACGAGGTGCTGCGTTA  CAACGCGAGCAACCAGTTCACCTGGCGTGTGGCGGCGAAAGATGTTGAAATGGGTGGCGTGCGTATCGAGGCGGGTCA  AACCCTGGCGCTGTTTCTGGGCAGCGCGAACCGTGACGCGAACATGTTCGAACGTCCGAACGACTTTGATCTGGACCGTC  CGAACAGCGCGCGTCACCTGAGCTTCGGTCAGGGCGTTCATGCGTGCCTGGCGGCGCAGCTGATCAGCCTGCAACTGAA  GTGGTTCTATGTGGCGCTGCTGAACCGTTTTCCGGGTATCCGTACCGCGGGCGAGCCGATTTGGAACGAAAACCTGGAGT  TTCGTAGCCTGCGTAGCCTGCCGCTGAGCCTGCGTGCGAAAAGCAAGAAAATCCCGCTGGGTGGCATTCCGAGCCCGAG  CACCGAACAGAGCGCGAAGAAAGTTCGTAAGAAAGCGGAGAACGCGCACAACACCCCGCTGCTGGTGCTGTACGGCAG  CAACATGGGTACCGCGGAAGGTACCGCGCGTGATCTGGCGGACATTGCGATGAGCAAGGGTTTCGCGCCGCAAGTTGCG  ACCCTGGATAGCCATGCGGGTAACCTGCCGCGTGAGGGTGCGGTGCTGATTGTTACCGCGAGCTATAACGGTCACCCGCC  GGACAACGCGAAGCAGTTTGTGGATTGGCTGGACCAAGCGAGCGCGGATGAAGTGAAAGGCGTTCGTTACAGCGTTTTC  GGTTGCGGCGACAAGAACTGGGCGACCACCTATCAGAAAGTGCCGGCGTTTATCGATGAAACCCTGGCGGCGAAGGGTG  CGGAAAACATTGCGGATCGTGGCGAGGCGGATGCGAGCGACGATTTCGAAGGTACCTACGAGGAATGGCGTGAGCACA  TGTGGAGCGACGTTGCGGCGTATTTTAACCTGGATATCGAAAACAGCGAGGACAACAAAAGCACCCTGAGCCTGCAGTTT  GTGGATAGCGCGGCGGACATGCCGCTGGCGAAGATGCACGGTGCGTTTAGCACCAACGTGGTTGCGAGCAAAGAACTG  CAACAACCGGGCAGCGCGCGTAGCACCCGTCACCTGGAAATCGAGCTGCCGAAAGAAGCGAGCTACCAAGAGGGTGAT  CACCTGGGCGTTATCCCGCGTAACTATGAAGGTATTGTGAACCGTGTTACCGCGCGTTTTGGCCTGGATGCGAGCCAGCA  AATTCGTCTGGAGGCGGAGGAAGAGAAGCTGGCGCACCTGCCGCTGGCGAAAACCGTGAGCGTTGAAGAGCTGCTGCA  GTACGTTGAGCTGCAAGACCCGGTTACCCGTACCCAACTGCGTGCGATGGCGGCGAAGACCGTTTGCCCGCCGCACAAA  GTGGAACTGGAGGCGCTGCTGGAAAAGCAGGCGTACAAAGAGCAAGTGCTGGCGAAGCGTCTGACCATGCTGGAACTG  CTGGAGAAGTATCCGGCGTGCGAAATGAAATTCAGCGAGTTTATCGCGCTGCTGCCGAGCATTCGTCCGCGTTACTATAG  CATCAGCAGCAGCCCGCGTGTTGACGAAAAGCAGGCGAGCATTACCGTGAGCGTGGTTAGCGGTGAAGCGTGGAGCGG  TTACGGCGAGTATAAAGGCATCGCGAGCAACTACCTGGCGGAACTGCAGGAAGGTGATACCATCACCTGCTTCATTAGCA  CCCCGCAAAGCGAATTTACCCTGCCGAAAGACCCGGAAACCCCGCTGATTATGGTTGGTCCGGGTACCGGTGTGGCGCCG  TTCCGTGGCTTTGTTCAGGCGCGTAAGCAACTGAAAGAACAGGGTCAAAGCCTGGGCGAGGCGCACCTGTACTTCGGTT  GCCGTAGCCCGCACGAGGATTACCTGTATCAGGAAGAGCTGGAAAACGCGCAAAGCGAGGGCATCATTACCCTGCACAC  CGCGTTTAGCCGTATGCCGAACCAGCCGAAGACCTATGTGCAGCACGTTATGGAACAAGATGGTAAGAAACTGATCGAG  CTGCTGGACCAGGGCGCGCACTTCTACATTTGCGGTGATGGTAGCCAAATGGCGCCGGCGGTGGAAGCGACCCTGATGA  AAAGCTATGCGGATGTGCACCAGGTTAGCGAGGCGGACGCGCGTCTGTGGCTGCAACAACTGGAAGAAAAGGGTCGTT ATGCGAAAGATGTGTGGGCGGGCTAAAAGCTT HindIII Figure S1. Codon optimised DNA sequence for His6-TxtE-BM3R overproduction in E. coli. Bases highlighted in green were used as restriction sites to clone into pET28a(+) to add an N-terminal His6tag. Bases highlighted in red were deleted using site directed mutagenesis to reduce the size of the linker between TxtE and BMR3 by 15aa.