Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2024 Jul 31.
Published in final edited form as: Methods Enzymol. 2023 Oct 12;693:31–49. doi: 10.1016/bs.mie.2023.09.007

P450-Catalyzed Atom Transfer Radical Cyclization

Heyu Chen 1,†, Wenzhen Fu 1,†, Yang Yang 1,2,*
PMCID: PMC11289761  NIHMSID: NIHMS2012119  PMID: 37977735

Abstract

Cytochromes P450 have been extensively studied in both fundamental enzymology and biotechnological applications. Over the past decade, by taking inspiration from synthetic organic chemistry, new classes of P450-catalyzed reactions that were not previously encountered in the biological world have been developed to address challenging problems in organic chemistry and asymmetric catalysis. In particular, by repurposing and evolving P450 enzymes, stereoselective biocatalytic atom transfer radical cyclization (ATRC) was developed as a new means to impose stereocontrol over transient free radical intermediates. In this chapter, we describe the detailed experimental protocol for the directed evolution of P450 atom transfer radical cyclases. We also delineate protocols for analytical and preparative scale biocatalytic atom transfer radical cyclization processes. These methods will find application in the development of new P450-catalyzed radical reactions, as well as other synthetically useful processes.

1. Introduction

Cytochromes P450 represent a unique family of heme-dependent enzymes found in all kingdoms of life responsible for oxidative transformations of metabolites and xenobiotics (Denisov et al., 2005; Meunier et al., 2004). Using the highly reactive iron oxo intermediate (Rittle & Green, 2010), P450 enzymes are capable of catalyzing a wide range of oxidative reactions (Montellano & Voss, 2002; Podust & Sherman, 2012), particularly C–H hydroxylation (Ortiz de Montellano, 2010), with excellent regio- and stereocontrol. In particular, the inherently promiscuous nature of P450 enzymes allows directed evolution to be easily performed, accommodating a range of synthetically valuable non-native substrates (Jung et al., 2011). In recent years, inspired by the structural similarity between the native Fe=O intermediate in P450 enzymology and synthetic Fe=C(R1)(R2) and Fe=NR intermediates, biocatalysis researchers repurposed P450 enzymes to catalyze carbene and nitrene transfer reactions that were never previously encountered in the biological world (Chen & Arnold, 2020; Yang & Arnold, 2021). The development of new-to-nature carbene and nitrene transferases significantly expanded the reaction space of P450 enzymes and provided powerful tools for asymmetric synthesis.

Recently, our group undertook a new approach to discover novel activities of P450 enzymes. Cognizant of the redox properties of the heme cofactor in P450s, we developed a class of P450-catalyzed atom transfer radical cyclization reactions using a metalloredox mechanism (Zhou et al., 2021; Y. Fu et al., 2022; W. Fu et al., 2023) (Fig. 1A). In our proposed catalytic cycle, starting from the ferrous enzyme, initial halogen atom transfer leads to a transient α-carbonyl radical and a ferric halide enzyme (Fig. 1B). The newly formed carbon-centered radical undergoes rapid cyclization with the pendant olefin, furnishing a new radical intermediate upon C–C bond formation. Finally, halogen atom rebound involving the enzymatic ferric halide species generates the atom transfer radical cyclization product and completes the catalytic cycle. This metalloredox-enabled atom transfer radical cyclization represents a new function of P450 enzymes, thereby further highlighting the synthetic potential of this useful enzyme family.

Fig. 1.

Fig. 1

Open in a new tab

P450-catalyzed atom transfer radical cyclization. A) Overall transformation; B) Proposed mechanism of P450-catalyzed atom transfer radical cyclization.

P450-catalyzed atom transfer radical cyclization affords a new strategy to impose stereocontrol over otherwise challenging free radical-mediated reactions. In the field of asymmetric catalysis, exerting diastereo- and enantiocontrol over radical intermediates has long been recognized as a difficult problem, in part due to the highly reactive nature of radical species and the lack of tight catalyst-substrate association (Sibi et al., 2003; Proctor et al., 2020; Mondal et al., 2022). In this regard, P450 atom transfer radical cyclases provide an alternative to develop otherwise challenging catalytic asymmetric radical reactions. P450 radical cyclases also complement recently developed ene reductase (ERED) and ketoreductase (KRED)-derived reductive radical photoenzymes (Emmanuel et al., 2023; Harrison et al., 2022), as overall redox-neutral radical reactions can be developed with P450 enzymes. In particular, the added halogen functional group resulting from halogen rebound allows convenient transformation of radical products. Furthermore, tertiary carbon-centered radicals could be conveniently generated and converted with evolved P450 radical cyclases, complementing ERED and KRED-catalyzed radical C–C bond formation that exhibited excellent efficiency and stereoselectivity with primary carbon-centered radicals (Emmanuel et al., 2016; Biegasiewicz et al., 2019; Black et al., 2020; Clayman & Hyster, 2020; Page et al., 2023, 2021; Gao et al., 2021; Nicholls et al., 2022; H. Fu et al., 2022, 2023; Huang et al., 2020, 2022; Zhang et al., 2023). In our recent mechanistic study, it was found that evolved P450 radical cyclases are bifunctional biocatalysts with a hydrogen bond donor residue playing a key role in enhancing enzyme activity and enantioselectivity (Y. Fu et al., 2022). This investigation highlights the versatility of directed evolution in discovering useful biocatalytic activation modes that are otherwise less straightforward to design. This redox-enabled new-to-nature enzymology could also be expanded to other heme proteins (Lubskyy et al., 2022).

In this chapter, we describe detailed procedures for the directed evolution of P450 radical cyclases as well as analytical and preparative scale reactions. Specifically, protocols for high-throughput protein engineering using 96-well plates, HPLC assays, and biocatalytic reactions on various representative scales are described below.

2. Materials

2.1. Cloning

  • Eppendorf Research® Plus single channel mechanical pipettes, variable volume

  • TempAssure® 0.2 mL PCR tubes, flat caps (USA Scientific, Catalog number: 1402–8100)

  • TOPQSC XK-400 Palm-Series Mini-Centrifuge

  • Bio-Rad T100™ Thermal Cycler

  • Bio-Rad PowerPac™ Basic power supply

  • Bio-Rad Mini-Sub® Cell GT

  • VWR® Blue Light Transilluminator

  • Nanodrop™ One Microvolume UV-Vis Spectrophotometer (Thermo Scientific™)

  • Bio-Rad MicroPulser™ electroporator

  • Fisherbrand® electroporation cuvettes-2mm gap

  • Gene of interest cloned (GOI) into pET-22b(+) vector between NdeI and XhoI with a C-terminal 6 × His-tag

  • Primers (005, 006, 007, 008, NDT, VHG, TGG, and rev primers; purchased from Integrated DNA Technologies or GENEWIZ)
    • 005 (forward primer for the amplification of GOI): 5’-GAAATAATTTTGTTTAACTTTAAGAAGGAGATATACATATG-3’
    • 006 (reverse primer for the amplification of GOI): 5’-GCCGGATCTCAGTGGTGGTGGTGGTGGTGCTCGAG-3’
    • 007 (reverse primer for the amplification of the backbone): reverse complement strand of 005
    • 008 (forward primer for the amplification of the backbone): reverse complement strand of 006
    • Forward (NDT, VHG, TGG) and reverse primers designed for site-saturation mutagenesis using the “22-c trick” method (Kille et al., 2013).
    • Example primers designed for site-saturation mutagenesis at the T327 site of parent variant “P”:
      • T327X_fwd_NDT: 5’- CGAAGCGCTGCGCTTATGGCCANDTGCTCCTGCGTTTTC-3’
      • T327X_fwd_VHG: 5’- CGAAGCGCTGCGCTTATGGCCAVHGGCTCCTGCGTTTTC-3’
      • T327X_fwd_TGG: 5’- CGAAGCGCTGCGCTTATGGCCATGGGCTCCTGCGTTTTC-3’
      • T327X_rev: 5’ -CCATAAGCGCAGCGCTTCGTTTAAGAC-3’

  • Backbone (ca. 100 ng/μL as determined by NanoDrop) of the pET-22b(+) vector; prepared by long-range PCR using pET-22b(+) as the template and 007 and 008 as primers.

  • Phusion® High-Fidelity DNA Polymerase, DMSO, Phusion® 5X HF Buffer, dNTPs, DpnI, from New England Biolabs (NEB)

  • PCR water (autoclaved MilliQ water)

  • 50X TAE buffer (50 mM EDTA, 2 M Tris, 1 M acetic acid)

  • 1% agarose gel (5 g agarose (low electroendosmosis, GOLDBIO, Catalog number: A-201–500) in 500 mL 1X TAE buffer, microwave until transparent)

  • 1 kb DNA ladder (NEB, Catalog number: N3232S)

  • Gel loading dye, purple (6X), no SDS (NEB, Catalog number: B7025S) with SYBR® Gold nucleic acid gel stain 10,000X concentrate in DMSO (Invitrogen, Catalog number: S11494)

  • Monarch® PCR & DNA Gel Extraction Kit (NEB)

  • Gibson Mastermix (Gibson et al., 2009) (320 μL 5X isothermal buffer (25% PEG-8000, 500 mM Tris-HCl pH 7.5, 50 mM MgCl2, 50 mM dithiothreitol, 1 mM each of the dNTPs, and 5 mM NAD)), 0.64 μL of 10 U/μL T5 exonuclease, 20 μL of 2 U/μL Phusion® High-Fidelity DNA Polymerase, 160 μL of 40 U/μL Taq DNA ligase from NEB)

  • Purified double-stranded DNA fragments with 18–22 bp overlaps

  • SOC medium (2% tryptone, 0.5% yeast extract, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl2, 10 mM MgSO4, and 20 mM glucose, autoclaved)

  • Electrocompetent E. coli strain E. cloni BL21(DE3) cells (Lucigen)

  • LBamp agar plate (15 g agar (molecular genetics grade, Fisher BioReagents™, Catalog number: BP1423–500) in Luria-Bertani medium with 0.1 mg/mL ampicillin sodium salt, Chem-Impex Int’l. Inc. Catalog number: 00516)

  • New Brunswick Innova 44R shakers

  • Microcentrifuge tubes (1.5 mL, 2 mL)

  • Eppendorf F570h ultra low temperature freezer

2.2. Enzyme expression in E. coli

  • Eppendorf Xplore® plus 12-channel pipettes, variable volumes

  • Thermo Scientific™ 1300 Series Class II, Type A2 Biological Safety Cabinet Packages, 120V 50/60 Hz (Catalog number: 1337)

  • LabGard® ES NU-425–600 Class II, Type A2 Biosafety Cabinet (NuAire)

  • Toothpicks (autoclaved)

  • EasyApp microporous film rolls, sterile (USA Scientific, Catalog number: 2977–6282)

  • Fisherbrand™ disposable sterile plastic culture tubes (Catalog number: FB149566B)

  • Erlenmeyer flasks

  • LBamp medium (Luria-Bertani medium with 0.1 mg/mL ampicillin)

  • HBamp medium (Hyperbroth (AthenaES) with 0.1 mg/mL ampicillin)

  • 0.5 M isopropyl β-D-1-thiogalactopyranoside (1000X IPTG) in MilliQ water (sterilized by membrane filtration through a 0.22-micron sterile cellulose acetate or PES filter)

  • 1.0 M 5-aminolevulinic acid (1000X ALA) in MilliQ water (sterilized by membrane filtration through a 0.22-micron sterile cellulose acetate or PES filter)

2.3. Whole-cell reaction

  • Eppendorf tabletop centrifuge 5910R

  • Eppendorf 5424R centrifuge

  • Thermo Scientific Sorvall Lynx 6000 superspeed centrifuge

  • Fishebrand microplate shaker

  • Corning LSE digital microplate shaker

  • Coy Lab vinyl anaerobic chamber

  • 50 mL centrifuge tubes

  • Biotage Isolera One Flash Chromatography System

  • SiliaFlash® P60, 40–63 μm (230–400 mesh), 60 Å (Silicycle, Catalog number: R12030B)

  • M9-N buffer (47.7 mM Na2HPO4, 22.0 mM KH2PO4, 8.6 mM NaCl, 2.0 mM MgSO4, and 0.1 mM CaCl2, pH 7.4)

  • 500 mM D-Glucose in M9-N buffer

  • Ethanol (200 Proof), Molecular Biology Grade (Fisher BioReagents™, Catalog number: BP28184)

  • 267 mM substrate in EtOH (200 Proof)

2.4. Normal phase HPLC analysis

  • Shimadzu i-series (66 MPa) HPLC

  • Chiralpak IG (4.6 mm × 25 cm, 5 micron) column

  • Vortex-Genie 2 Mixer (Fisherbrand®, Catalog number: 12–812)

  • Reagent alcohol (90% EtOH, HPLC grade, Fisher Chemical™, Catalog number: A9954)

  • Hexanes (98.5% hexane, mixture of isomers, HPLC grade, Sigma-Aldrich, Catalog number: 293253)

  • Extraction solution (1 mM mesitylene in 1:1 (v/v) EtOAc/hexanes)

2.5. Pyridine hemochromagen assay

  • Shimadzu UV-1800 spectrophotometer

  • Plastic cuvettes

  • Solution I (20 mL pyridine, 20 mL 0.5 M NaOH, 250 L 0.1 M K3[Fe(CN)6], 10 mL MilliQ water)

  • Solution III (0.1 g/mL Na2S2O4 in 0.1 M NaOH, freshly prepared)

3. Protocols

3.1. Cloning for a site-saturated mutagenesis screening library

  1. Prepare the primer mix:
    • Forward primer mix: dilute 24 μL T327X_fwd_NDT (100 μM), 18 μL T327X_fwd_VHG (100 μM), and 2 μL T327X_fwd_TGG (100 μM) with 396 μL PCR H2O in a 1.5 mL microcentrifuge tube, vortex and mix it well to make the Fwd-primer mix with a final concentration of 10 μM.
    • Reverse primer mix: dilute 22 μL T327X _rev (100 μM) with 198 μL PCR H2O in a 1.5 mL microcentrifuge tube, vortex, and mix it well to make the Rev-primer mix with a final concentration of 10 μM.
  2. Set up the PCR on ice:
    • PCR1: Transfer 13.5 μL PCR H2O, 5 μL 5X HF buffer, 1 μL DMSO, 1 μL 10 mM dNTPs, 1 μL DNA template (30–150 ng/μL), 1.5 μL Fwd-primer mix, and 1.5 μL 006 primer to a PCR tube, mix it by tapping gently and quickly spin it down using the minifuge (turn the minifuge on and immediately off).
    • PCR2: Transfer 13.5 μL PCR H2O, 5 μL 5X HF buffer, 1 μL DMSO, 1 μL 10 mM dNTPs, 1 μL DNA template (30–150 ng/μL), 1.5 μL Rev-primer mix, and 1.5 μL 005 primer to a PCR tube, mix it by tapping gently and quickly spin it down using the minifuge (turn the minifuge on and immediately off).
    • Add 0.5 μL Phusion polymerase to PCR1 and PCR2, mix it by tapping gently, and quickly spin it down using the minifuge (turn the minifuge on and immediately off).

  3. Set up the thermocycler’s parameters: Initial denaturation step (98 °C, 30 s), Denaturation step (98 °C, 10 s), Annealing step (55 °C, 10 s), Extension step (72 °C, 30 s), cycles (30–35 cycles), Final extension step (72 °C, 10 min), and hold at 12 °C. (For Phusion polymerase, the extension rate is 1000 bp/(20–30) s. Calculate the time required for the extension step based on the length of your DNA of interest and the extension rate of Phusion polymerase.)

  4. Place sealed PCR tubes in the thermocycler and start the program.

  5. Run the DNA gel electrophoresis to purify the PCR product:
    • Cast the 1% Agarose gel in 1X TAE buffer.
    • When the PCR completes, add 5 μL 6X DNA loading dye supplemented with SYBE gold (0.2 μL/mL) to each PCR tube. Ensure good mixing by pipetting up and down.
    • Load the PCR product to the agarose gel.
    • Load 1kb DNA ladder to the agarose gel.
    • Start DNA gel electrophoresis (150 mV, 20 min).
    • Isolate the agarose gel containing the DNA product with the aid of a blue light transilluminator.
    • Isolate the DNA product with NEB’s Monarch DNA Gel Extraction Kit; usually 6 μL elution buffer is used for each PCR DNA to ensure a relatively high final concentration of DNA products.
    • Measure the DNA concentration using Nanodrop and adjust the concentration to 100 ng/μL with elution buffer.
    • Keep the DNA products in a −20 °C freezer for storage.
  6. Set up the Gibson Assembly:
    • Transfer 5 μL Gibson Mastermix, 1.2 μL backbone, 0.6 μL DNA fragment 1 (from PCR1), 0.6 μL DNA fragment 2 (from PCR2), and 2.6 μL PCR H2O into a PCR tube.
    • Tap the PCR tube gently with fingers and quickly spin it down using a minifuge (turn the minifuge on and immediately off).
    • Incubate the Gibson mixture at 50 °C for 60 min with the thermocycler.
    • Keep the Gibson mixture on ice for transformation. Remaining Gibson product is kept in a −20 °C freezer for storage.
  7. Set up the transformation:
    • Thaw the SOC media.
    • Thaw a tube of electrocompetent E. coli cells (50 μL) from the −80 °C freezer. Once thawed, electrocompetent cells are kept on ice.
    • Cool the electroporation cuvette on ice.
    • Add 1 μL the above Gibson product to thawed electrocompetent E. coli cells using a 2.5 μL pipette.
    • Quickly transfer the electrocompetent E. coli cells containing the Gibson product to an electroporation cuvette.
    • Wipe the electrodes of the cuvette with Kimwipe and apply electroporation with the “Bacteria” method (1.8 kV).
    • Quickly transfer 750 μL SOC media to the cuvette and mix by pipetting up and down.
    • Incubate the SOC culture at 37 °C, 230 rpm for 45 min in a New Brunswick Innova 44R shaker.
    • Inoculate 30–100 μL SOC culture on an LB Agar (Amp) plate and incubate the Agar plate at 37 °C for 10–12 h.
    • Keep the remaining SOC culture in a 4 °C fridge. This SOC culture is usually good for plating for approximately one week.

  8. Parent control is required for screening in 96-well plates. Freshly transformed E. coli cells carrying the plasmid encoding the parent enzyme are preferred to ensure good reproducibility.

3.2. High-throughput experimentation in 96-well plates:

  1. Grow the overnight culture in 96-well plates:
    • Transfer 400 μL LBamp media to each well in a 96-well plate using an Eppendorf Xplorer 12-channel pipette.
    • Pick single colonies for the parent enzyme with autoclaved toothpicks and add them to the A1, B2, C3, D4, E5, F6, G7, and H8 wells.
    • Pick single colonies from the agar plate with the SSM screening library with autoclaved toothpicks and add one single colony to each well except A1, B2, C3, D4, E5, F6, G7, and H8.
    • Remove the toothpicks.
    • Cover the 96-well plate with a microporous film.
    • Incubate starter cultures in the 96-well plate at 37 °C, 250 rpm for 12–14 h in a New Brunswick Innova 44R shaker.
  2. Protein expression in a 96-well plate:
    • Transfer 950 μL HBamp media to each well in a new sterilized 96-well plate with an Eppendorf Xplorer 12-channel pipette.
    • Transfer 50 μL LBamp overnight culture to each well of the 96-well plate containing HBamp media with a 12-channel pipette; change the tips each time.
    • Cover the 96-well plate with a microporous film.
    • Incubate the 96-well plate at 37 °C, 250 rpm for 2.5 h in a New Brunswick Innova 44R shaker until OD600 reaches ca. 2.
    • Transfer 100 μL sterile 50% glycerol to each well of a separate 96-well microplate for glycerol stock storage.
    • Transfer 100 μL LBamp culture from the starter culture plate to each well of this microplate.
    • Cover the lid and mix it by gentle swirling.
    • Keep the bacterial glycerol stock in the microplate in a −80 °C freezer.
    • Thaw the 1000X IPTG and 1000X ALA stock from the −20 °C freezer.
    • After 2.5 h, put the 96-well plate with expression culture on ice for 20 min.
    • Prepare the IPTG/ALA stock solution by mixing 20 mL HBamp media with 400 μL 1000X IPTG and 400 μL 1000X ALA stock solutions.
    • Transfer 50 μL of this newly prepared, diluted IPTG/ALA solution to each well of the 96-well plate containing expression cultures using an Eppendorf Xplorer 12-channel pipette.
    • Gently shake the plate to ensure good mixing.
    • Re-cover the 96-well plate with the microporous film.
    • Incubate the expression culture at 22 °C, 220 rpm for 22 h in a New Brunswick Innova 44R shaker.
  3. Set up screening reactions with the whole E. coli cells in 96-well plate:
    • Cool the Eppendorf tabletop centrifuge 5910R to 4 °C (takes 30 min).
    • Put the 96-well plate with HBamp culture on ice.
    • Spin down the cell pellets at 3000 rpm at 4 °C for 3 min and discard the supernatant.
    • Transfer 345 μL M9-N buffer and 40 μL D-Glucose solution (0.5 M in M9-N buffer) to each well and shake the plate in a Fisher Scientific microplate shaker at 800 rpm for resuspension.
    • Transfer the 96-well plate and substrate stock to a Coy anaerobic chamber.
    • Add 15 μL substrate stock solution (4 μmol, 0.27 M in EtOH, 200 proof) to each well using an Eppendorf Xplorer 12-channel pipette to start the reactions. Typically, there is no need to change pipette tips at this stage.
    • Cover the plate with resealable aluminum foil.
    • Shake the plate at 680 rpm for 12 h on a Corning digital microplate shaker.
  4. Reactions work-up:
    • Add 600 μL extraction solution to each well to quench the reaction.
    • Cover the plate with a resealable silicon mat.
    • Shake the plate vigorously for at least 30 s to ensure good extraction.
    • Put the plate in the Eppendorf tabletop centrifuge 5910R and centrifuge for 5 min at 4500 rpm to separate the organic and aqueous layers.
    • Transfer 360 μL of the organic phase using a 12-channel pipette to clean inserts (500 μL).
    • Put the inserts containing the organic layer into the empty 2 mL sample vials and close the caps of these sample vials. Good sealing of sample vials can minimize the evaporation of organic solvents.
    • Keep the sample vials at 4 °C until chromatographic analysis.
    • Analyze the organic phase using normal phase chiral HPLC (Shimadzu i-series (66 MPa) HPLC, Chiralpak IG, isocratic elution: 30% EtOH/hexanes, 9 min)
    • Identify hits from high-throughput screening.

3.3. Analytical scale reactions to validate the screening hits:

  1. Grow the overnight culture in culture tubes:
    • Transfer 4.5 mL LBamp medium to a culture tube.
    • Inoculate LBamp with bacteria glycerol stock using sterile toothpicks.
    • Loosely cap the culture tube to ensure good aeration.
    • Incubate the starter culture at 37 °C, 230 rpm for 12–14 h in an Eppendorf Innova 44R shaker.
  2. Protein expression in 125 mL Erlenmeyer flasks:
    • To validate hits from high-throughput screening, a copy of bacterial glycerol stock is usually saved. The hits are also sequenced by Sanger sequencing.
    • Transfer 28.5 mL HBamp medium to a 125 mL Erlenmeyer flask.
    • Inoculate the HBamp medium with 1.5 mL overnight culture.
    • Incubate at 37 °C, 230 rpm in an Eppendorf Innova 44R shaker until OD600 reaches ca. 2 (approximately 2 h).
    • Thaw the 1000X IPTG and 1000X ALA stock from the −20 °C freezer in water.
    • After 2 h, put the Erlenmeyer flask on ice for 20 min.
    • Transfer 30 μL IPTG/ALA stock solution (vide supra) to each flask.
    • Incubate the expression culture at 22 °C, 150 rpm for 22 h in an Eppendorf Innova 44R shaker.
  3. Set up validation reactions with the whole E. coli cells in 2 mL sample vials
    • Set the temperature of Eppendorf 5910R tabletop centrifuge to 4 °C ahead of time.
    • Transfer the expression culture to 50 mL centrifuge tubes.
    • Spin down the cell pellets at 3000 rpm at 4 °C for 3 min.
    • Discard the supernatant.
    • Add M9-N buffer to normalize the OD600 to 30 ± 1.
    • Shake the tube in a Fisher Scientific microplate shaker at 800 rpm to resuspend the cell pellet in M9-N buffer.
    • Use 2 mL cell suspension for pyridine hemochromagen assay to determine heme concentration.
    • Analytical scale reactions are usually carried out in triplicate.
    • Transfer 345 μL of the cell suspension to 2.0 mL sample vials.
    • Transfer the sample vials, D-Glucose stock solution, and substrate stock solutions to a Coy anaerobic chamber.
    • Add 40 μL D-Glucose stock to each sample vial.
    • Add 15 μL substrate stock solution (4 μmol, 0.27 M in EtOH, 200 proof) to each vial.
    • Quickly cap the vials and immediately start shaking on a Corning digital microplate shaker at 680 rpm for 12–24 h.
  4. Pyridine hemochromagen assay (Berry & Trumpower, 1987):
    • Lyse the cell (on ice) with BioLogics ultrasonic homogenizer (model 150VT) equipped with a stepped microtip (3 min, 2 cycles, 1 sec on, 1 sec off, 40% amplitude).
    • Centrifuge the lysate at 15000 rpm, 10 min, 4 °C in an Eppendorf 5424 R centrifuge.
    • Mix 500 μL supernatant (clarified lysate) with 500 μL Solution I in a plastic cuvette.
    • Add 10 μL Solution III to each cuvette, mix by pipetting.
    • Acquire UV-Visible absorbance spectrum to determine the heme b concentration (Shimadzu UV-1800, ε557nm = 34.7 mM−1 cm−1, baseline correction at 600 nm, blank: 500 μL M9-N buffer and 500 μL Solution I)
  5. Reactions work-up:
    • Add 600 μL extraction solution to each vial to quench the reaction.
    • Vortex each vial for at least 10 s to ensure good mixing.
    • Transfer the mixture to 1.5 mL centrifuge tubes.
    • Centrifuge at 15000 rpm for 5 min in an Eppendorf 5424 R centrifuge to separate the organic and the aqueous layers.
    • Transfer 360 μL of the organic layer to a 0.5 mL insert and place these inserts in sample vials for HPLC analysis.
    • Keep the sample vials at 4 °C for further chromatography screening.
    • Analyze the organic phase using normal phase chiral HPLC (Shimadzu i-series (66 MPa) HPLC, Chiralpak IG, isocratic elution: 30% EtOH/hexanes, 9 min)

3.4. Preparative-scale reactions:

  1. Grow the overnight culture in a 125 mL Erlenmeyer flask:
    • Transfer 15 mL LBamp medium to a 125 mL Erlenmeyer flask.
    • Inoculate the medium with bacteria glycerol stock using a sterile toothpick.
    • Incubate the starter culture at 37 °C, 230 rpm for 12–14 h in an Eppendorf Innova 44R shaker.
  2. Protein expression in a 4 L Erlenmeyer flask:
    • Inoculate 1 L HBamp medium in a 4 L Erlenmeyer flask with 10 mL overnight culture.
    • Incubate at 37 °C, 230 rpm in an Eppendorf Innova 44R shaker until OD600 reaches 2 (ca. 3.5 h).
    • After 3.5 h, put the Erlenmeyer flask on ice for 30 min.
    • Thaw the 1000X IPTG stock and 1000X ALA stock.
    • Transfer 1 mL IPTG/ALA stock solution to each flask.
    • Incubate the expression culture at 22 °C, 150 rpm for 20 h in an Eppendorf Innova 44R shaker.
  3. Set up preparative-scale reactions with whole E. coli cells in 1 L Erlenmeyer flask:
    • Pellet the cells by centrifugation at 3,000 g, 4 °C for 5 min using a Thermo Scientific Sorvall Lynx 6000 superspeed centrifuge.
    • Add M9-N buffer to resuspend the cell (OD600 = 30–40).
    • Transfer the cell suspension to a 1 L Erlenmeyer flask with a screw cap and keep it on ice until further use.
    • Use 2 mL cell suspension for pyridine hemochromagen assay to determine heme concentration (as described in Section 3.3).
    • Transfer the Erlenmeyer flask, D-Glucose stock, and substrate stock to a Coy anaerobic chamber.
    • Add 35 mL D-Glucose stock solution and 7 mL substrate (430 mM in EtOH, 200 proof) to the flask.
    • Cap the flask and seal it with Parafilm.
    • Take the flask out of the Coy Chamber.
    • Shake the flask at 25 °C, 200 rpm for 7–18 h in an Eppendorf Innova 44R shaker.
  4. Reaction work-up:
    • Extract the reaction mixture with EtOAc.
    • Centrifuge the mixture at 15000 g, 4 °C for 30 min using a Thermo Scientific Sorvall Lynx 6000 superspeed centrifuge.
    • Extract the aqueous layer with EtOAc for a total of three times.
    • Dry the combined organic phase over MgSO4.
    • Filter the combined organic through a celite plug to remove MgSO4.
    • Concentrate the organic phase via rotary evaporation.
    • Purify the compound by silica gel column chromatography with the aid of a Biotage Isolera.

3.5. HPLC calibration curves:

  • Prepare a 100 mM stock solution of authentic products in EtOAc.

  • Add 400 μL M9-N buffer and 600 μL extraction solution to a 1.5 mL microcentrifuge tube.

  • Add an aliquot (3, 6, 12, 24, 48 μL product stock solution, respectively) to 1.5 mL microcentrifuge tubes.

  • Vortex the mixture (20 s for three times).

  • Centrifuge at 15,000 rpm for 5 min in an Eppendorf 5424 R centrifuge.

  • Transfer 360 μL of the organic phase to 0.5 mL inserts in the sample vials.

  • Analyze the organic phase using normal phase chiral HPLC (Shimadzu i-series (66 MPa) HPLC, Chiralpak IG, isocratic elution: 30% EtOH/hexanes, 9 min)

4. Summary

By drawing inspirations from synthetic organic and organometallic chemistry, a range of new enzyme functions were discovered in recently years to significantly expand the reaction space of natural enzymes. Advances in high-throughput screening methods, along with directed evolution and protein engineering, accelerate the design and development of enzymes with unnatural activities. In this chapter, we described detailed methods to perform P450 enzyme-catalyzed atom transfer radical cyclization reactions. Protocols for the high-throughput P450 enzyme engineering in 96-well plates, HPLC screening assays, analytical and large-scale biocatalytic reactions were provided. These protocols may find use in the further development of P450 radical cyclases and other useful activities of P450 enzymes.

Acknowledgments

We acknowledge the NIH (R35GM147387) and ACS PRF (65807-DNI1) for financial support.

References

  1. Berry EA, & Trumpower BL (1987). Simultaneous determination of hemes a, b, and c from pyridine hemochrome spectra. Analytical Biochemistry, 161(1), 1–15. 10.1016/0003-2697(87)90643-9 [DOI] [PubMed] [Google Scholar]
  2. Biegasiewicz KF, Cooper SJ, Gao X, Oblinsky DG, Kim JH, Garfinkle SE, Joyce LA, Sandoval BA, Scholes GD, & Hyster TK (2019). Photoexcitation of flavoenzymes enables a stereoselective radical cyclization. Science, 364(6446), 1166–1169. 10.1126/science.aaw1143 [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Black MJ, Biegasiewicz KF, Meichan AJ, Oblinsky DG, Kudisch B, Scholes GD, & Hyster TK (2020). Asymmetric redox-neutral radical cyclization catalysed by flavin-dependent ‘ene’-reductases. Nature Chemistry, 12(1), 71–75. 10.1038/s41557-019-0370-2 [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Chen K, & Arnold FH (2020). Engineering new catalytic activities in enzymes. Nature Catalysis, 3(3), 203–213. 10.1038/s41929-019-0385-5 [DOI] [Google Scholar]
  5. Clayman PD, & Hyster TK (2020). Photoenzymatic Generation of Unstabilized Alkyl Radicals: An Asymmetric Reductive Cyclization. Journal of the American Chemical Society, 142(37), 15673–15677. 10.1021/jacs.0c07918 [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Denisov IG, Makris TM, Sligar SG, & Schlichting I (2005). Structure and Chemistry of Cytochrome P450. Chemical Reviews, 105(6), 2253–2278. 10.1021/cr0307143 [DOI] [PubMed] [Google Scholar]
  7. Emmanuel MA, Bender SG, Bilodeau C, Carceller JM, DeHovitz JS, Fu H, Liu Y, Nicholls BT, Ouyang Y, Page CG, Qiao T, Raps FC, Sorigué DR, Sun S-Z, Turek-Herman J, Ye Y, Rivas-Souchet A, Cao J, & Hyster TK (2023). Photobiocatalytic Strategies for Organic Synthesis. Chemical Reviews, 123(9), 5459–5520. 10.1021/acs.chemrev.2c00767 [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Emmanuel MA, Greenberg NR, Oblinsky DG, & Hyster TK (2016). Accessing non-natural reactivity by irradiating nicotinamide-dependent enzymes with light. Nature, 540(7633), 414–417. 10.1038/nature20569 [DOI] [PubMed] [Google Scholar]
  9. Fu H, Cao J, Qiao T, Qi Y, Charnock SJ, Garfinkle S, & Hyster TK (2022). An asymmetric sp3–sp3 cross-electrophile coupling using ‘ene’-reductases. Nature, 610(7931), 302–307. 10.1038/s41586-022-05167-1 [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Fu H, Qiao T, Carceller JM, MacMillan SN, & Hyster TK (2023). Asymmetric C-Alkylation of Nitroalkanes via Enzymatic Photoredox Catalysis. Journal of the American Chemical Society, 145(2), 787–793. 10.1021/jacs.2c12197 [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Fu W, Neris NM, Fu Y, Zhao Y, Krohn-Hansen B, Liu P, & Yang Y (2023). Enzyme-controlled stereoselective radical cyclization to arenes enabled by metalloredox biocatalysis. Nature Catalysis, 6(7), 628–636. 10.1038/s41929-023-00986-5 [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Fu Y, Chen H, Fu W, Garcia-Borràs M, Yang Y, & Liu P (2022). Engineered P450 Atom-Transfer Radical Cyclases are Bifunctional Biocatalysts: Reaction Mechanism and Origin of Enantioselectivity. Journal of the American Chemical Society, 144(29), 13344–13355. 10.1021/jacs.2c04937 [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Gao X, Turek-Herman JR, Choi YJ, Cohen RD, & Hyster TK (2021). Photoenzymatic Synthesis of α-Tertiary Amines by Engineered Flavin-Dependent “Ene”-Reductases. Journal of the American Chemical Society, 143(47), 19643–19647. 10.1021/jacs.1c09828 [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Gibson DG, Young L, Chuang R-Y, Venter JC, Hutchison CA, & Smith HO (2009). Enzymatic assembly of DNA molecules up to several hundred kilobases. Nature Methods, 6(5), 343–345. 10.1038/nmeth.1318 [DOI] [PubMed] [Google Scholar]
  15. Harrison W, Huang X, & Zhao H (2022). Photobiocatalysis for Abiological Transformations. Accounts of Chemical Research, 55(8), 1087–1096. 10.1021/acs.accounts.1c00719 [DOI] [PubMed] [Google Scholar]
  16. Huang X, Feng J, Cui J, Jiang G, Harrison W, Zang X, Zhou J, Wang B, & Zhao H (2022). Photoinduced chemomimetic biocatalysis for enantioselective intermolecular radical conjugate addition. Nature Catalysis, 5(7), 586–593. 10.1038/s41929-022-00777-4 [DOI] [Google Scholar]
  17. Huang X, Wang B, Wang Y, Jiang G, Feng J, & Zhao H (2020). Photoenzymatic enantioselective intermolecular radical hydroalkylation. Nature, 584(7819), 69–74. 10.1038/s41586-020-2406-6 [DOI] [PubMed] [Google Scholar]
  18. Jung ST, Lauchli R, & Arnold FH (2011). Cytochrome P450: Taming a wild type enzyme. Current Opinion in Biotechnology, 22(6), 809–817. 10.1016/j.copbio.2011.02.008 [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Kille S, Acevedo-Rocha CG, Parra LP, Zhang Z-G, Opperman DJ, Reetz MT, & Acevedo JP (2013). Reducing Codon Redundancy and Screening Effort of Combinatorial Protein Libraries Created by Saturation Mutagenesis. ACS Synthetic Biology, 2(2), 83–92. 10.1021/sb300037w [DOI] [PubMed] [Google Scholar]
  20. Lubskyy A, Guo C, Chadwick RJ, Petri-Fink A, Bruns N, & Pellizzoni MM (2022). Engineered myoglobin as a catalyst for atom transfer radical cyclisation. Chemical Communications, 58(78), 10989–10992. 10.1039/D2CC03227A [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Meunier B, de Visser SP, & Shaik S (2004). Mechanism of Oxidation Reactions Catalyzed by Cytochrome P450 Enzymes. Chemical Reviews, 104(9), 3947–3980. 10.1021/cr020443g [DOI] [PubMed] [Google Scholar]
  22. Mondal S, Dumur F, Gigmes D, Sibi MP, Bertrand MP, & Nechab M (2022). Enantioselective Radical Reactions Using Chiral Catalysts. Chemical Reviews, 122(6), 5842–5976. 10.1021/acs.chemrev.1c00582 [DOI] [PubMed] [Google Scholar]
  23. Montellano P. R. O. de, & Voss JJD (2002). Oxidizing species in the mechanism of cytochrome P450. Natural Product Reports, 19(4), 477–493. 10.1039/B101297P [DOI] [PubMed] [Google Scholar]
  24. Nicholls BT, Oblinsky DG, Kurtoic SI, Grosheva D, Ye Y, Scholes GD, & Hyster TK (2022). Engineering a Non-Natural Photoenzyme for Improved Photon Efficiency**. Angewandte Chemie International Edition, 61(2), e202113842. 10.1002/anie.202113842 [DOI] [PubMed] [Google Scholar]
  25. Ortiz de Montellano PR (2010). Hydrocarbon Hydroxylation by Cytochrome P450 Enzymes. Chemical Reviews, 110(2), 932–948. 10.1021/cr9002193 [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Page CG, Cao J, Oblinsky DG, MacMillan SN, Dahagam S, Lloyd RM, Charnock SJ, Scholes GD, & Hyster TK (2023). Regioselective Radical Alkylation of Arenes Using Evolved Photoenzymes. Journal of the American Chemical Society, 145(21), 11866–11874. 10.1021/jacs.3c03607 [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Page CG, Cooper SJ, DeHovitz JS, Oblinsky DG, Biegasiewicz KF, Antropow AH, Armbrust KW, Ellis JM, Hamann LG, Horn EJ, Oberg KM, Scholes GD, & Hyster TK (2021). Quaternary Charge-Transfer Complex Enables Photoenzymatic Intermolecular Hydroalkylation of Olefins. Journal of the American Chemical Society, 143(1), 97–102. 10.1021/jacs.0c11462 [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Podust LM, & Sherman DH (2012). Diversity of P450 enzymes in the biosynthesis of natural products. Natural Product Reports, 29(10), 1251–1266. 10.1039/C2NP20020A [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Proctor RSJ, Colgan AC, & Phipps RJ (2020). Exploiting attractive non-covalent interactions for the enantioselective catalysis of reactions involving radical intermediates. Nature Chemistry, 12(11), 990–1004. 10.1038/s41557-020-00561-6 [DOI] [PubMed] [Google Scholar]
  30. Rittle J, & Green MT (2010). Cytochrome P450 Compound I: Capture, Characterization, and C-H Bond Activation Kinetics. Science, 330(6006), 933–937. 10.1126/science.1193478 [DOI] [PubMed] [Google Scholar]
  31. Sibi MP, Manyem S, & Zimmerman J (2003). Enantioselective Radical Processes. Chemical Reviews, 103(8), 3263–3296. 10.1021/cr020044l [DOI] [PubMed] [Google Scholar]
  32. Yang Y, & Arnold FH (2021). Navigating the Unnatural Reaction Space: Directed Evolution of Heme Proteins for Selective Carbene and Nitrene Transfer. Accounts of Chemical Research, 54(5), 1209–1225. 10.1021/acs.accounts.0c00591 [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Zhang Z, Feng J, Yang C, Cui H, Harrison W, Zhong D, Wang B, & Zhao H (2023). Photoenzymatic enantioselective intermolecular radical hydroamination. Nature Catalysis. 10.1038/s41929-023-00994-5 [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Zhou Q, Chin M, Fu Y, Liu P, & Yang Y (2021). Stereodivergent atom-transfer radical cyclization by engineered cytochromes P450. Science, 374(6575), 1612–1616. 10.1126/science.abk1603 [DOI] [PMC free article] [PubMed] [Google Scholar]

RESOURCES