For controlled oxidation of 2, the reactions were performed as follows: To
obtain the predominant product S-2a, the reaction mixture containing 10 μM
CreHI, 10 μM CreJEF, 20 mM MgCl2, 2 mM MnCl2, 2 mM ATP, and 2 mM
NADPH was first incubated at 30 °C for 2 h. Following this initial incubation,
CreD (10 μM) was added into the mixture and allowed to incubate for an
additional 2 h. To produce 2b as the major product, CreG (10 μM) with its
cofactor NAD+ (2 mM) was added into the above post-CreD reaction mix-
ture, followed by another 2-h incubation. To predominantly produce 2c, the
reaction mixture containing 10 μM CreHI, 10 μM CreJEF, 10 μM CreG, 20 mM
MgCl2, 2 mM MnCl2, 2 mM ATP, 2 mM NAD+, and 3 mM NADPH was first
incubated at 30 °C for 2 h. Subsequently, an additional batch of CreJEF (final
concentration of 20 μM, including the first addition) and NADPH (final
concentration of 6 mM, including the first addition) were added into the
above mixture, followed by another 2-h incubation. Finally, CreD (10 μM)
was added into the mixture and followed by incubation at 30 °C for 2 h.
Methods
Crystallization, Data Collection, and Structure Determination. Before crystal-
lization, the protein was diluted to 0.3 mM by mixing with 10 mM Tris·HCl
(pH 8.0), supplemented with 12 mM substrate 1′ or 2′. Crystallization conditions
were determined using commercial high throughput screening kits pur-
chased from Hampton Research and a nanoliter drop-setting Mosquito
robot (TTP Labtech) operating with 96-well plates. The crystallization
conditions amenable to crystal generations were further optimized in
24-well crystallization plates. The crystals of CreJ in complex with 1′ used for
X-ray data collection were obtained in hanging drops containing 0.2 M
ammonium sulfate, 0.1 M sodium cacodylate trihydrate, pH 6.4, and 27%
PEG8000, whereas the crystals of CreJ in complex with 2′ were obtained in
0.2 M sodium iodide and 20% PEG3350. The data were collected at the
Shanghai Synchrotron Radiation Facility, Beamline BL17U (53), in a 100-K
nitrogen stream. Data indexing, integration, and scaling were conducted
using MOSFLM (54). The CreJ structure was determined by molecular re-
placement using CCP4 program suite (55) and the PDB file 4RM4 (56) as a
search model. The structure was automatically built by ARP/wARP in the
CCP4i program package (57). Refinement of the structure was performed
using the programs COOT (58) and PHENIX (59). The final model was eval-
uated using PROCHECK (60). All molecular graphics were created using
PyMOL v1.8 (Schrödinger).
Analytical Methods. Samples were analyzed on an Agilent 1260 infinity HPLC
system (Agilent Technologies) with a photodiode array detector. Reaction
mixtures were separated on a ZORBAX SB-C18 column (Agilent Technologies)
by using a linear mobile phase gradient ranging from 2% (vol/vol) acetonitrile
in 0.1% (vol/vol) TFA aqueous solution to 80% (vol/vol) acetonitrile in 0.1%
(vol/vol) TFA aqueous solution over 25 min. The flow rate was set to 1 mL/min
and the injection volume was 20 μL. The detection wavelength was set to
275 nm. Substrate consumption and product formation were quantified by
HPLC peak area integration using corresponding authentic compounds as
standards. Product structural assignments were performed by comparison of
a detected compound with the corresponding authentic standard regarding
retention time and coinjection confirmation, HRMS, and/or NMR. HRMS data
were recorded in the negative and/or positive ionization mode on a MaXis
Impact UHR-qTOF System (Bruker).
Enzymatic Assays. Unless specified otherwise, all enzymatic assays were car-
ried out in 100 μL of 50 mM Tris·HCl buffer (pH 8.0) at 30 °C for 2 h, and the
final concentration of each enzyme was 10 μM. Methanol (3× volume) was
added to quench reactions. Precipitated proteins were removed by centri-
fugation at 20,000 × g for 10 min and the supernatants were used as LC-MS
samples. For reactions catalyzed by CreHI, the reaction mixture contained
1 mM substrate, 20 mM MgCl2, 2 mM MnCl2, 2 mM ATP, and purified CreH
and CreI. For CreJEF activity toward phosphorylated compounds (2′–9′), the
CreHI reactions were used to generate the phosphorylated substrates. Next,
CreJ, CreE, and CreF together with 3 mM NADPH were added into the in-
dividual post-CreHI reaction mixture.
ACKNOWLEDGMENTS. We thank the staff of BL17U Beamline at the Shanghai
Synchrotron Radiation Facility and Dr. Shilong Fan (Tsinghua University)
for assistance during X-ray diffraction data collection and Dr. Jeffrey D.
Kittendorf (Alluvium Biosciences) for helpful discussion and proofreading
of the manuscript. This work was supported by the National Natural Science
Foundation of China under Grant NSFC 31422002, Shandong Provincial Nat-
ural Science Foundation Grant JQ201407, the Key Frontier Project of the
Chinese Academy of Sciences, QYZDB-SSW-SMC042 (to S.L.), the 973 Project
from Ministry of Science and Technology Grant 2012CB7211-04 (to S.-J.L.),
and Grant NSFC 31270784 from the National Natural Science Foundation of
China (to Y.F.).
For one-pot chemomimetic alkylphenol oxidation reactions, the reaction
mixtures contained CreHI, CreJEF, and the two optional enzymes CreG (with
2 mM NAD+) and CreC (with 2 mM NADP+), 1 mM substrate 2–7, and nec-
essary cofactors, including 20 mM MgCl2, 2 mM MnCl2, 2 mM ATP, and 3 mM
NADPH. After incubation at 30 °C for 2 h, CreD was added into each of the
one-pot mixtures, followed by another 2-h incubation at 30 °C.
1. Fiege H, et al. (2000) Phenol Derivatives. Ullmann’s Encyclopedia of Industrial
Chemistry (Wiley, Hoboken, NJ).
2. Bergé A, et al. (2012) Meta-analysis of environmental contamination by alkylphenols.
Environ Sci Pollut Res Int 19:3798–3819.
3. Ying GG, Williams B, Kookana R (2002) Environmental fate of alkylphenols and al-
kylphenol ethoxylates: A review. Environ Int 28:215–226.
16. Kille S, Zilly FE, Acevedo JP, Reetz MT (2011) Regio- and stereoselectivity of P450-
catalysed hydroxylation of steroids controlled by laboratory evolution. Nat Chem 3:
738–743.
17. Kolev JN, Zaengle JM, Ravikumar R, Fasan R (2014) Enhancing the efficiency and re-
gioselectivity of P450 oxidation catalysts by unnatural amino acid mutagenesis.
ChemBioChem 15:1001–1010.
4. Hudlicky
M (1990) Oxidations in Organic Chemistry (American Chemical Society,
18. Zhang K, Shafer BM, Demars MD, 2nd, Stern HA, Fasan R (2012) Controlled oxidation
of remote sp3 C–H bonds in artemisinin via P450 catalysts with fine-tuned regio- and
stereoselectivity. J Am Chem Soc 134:18695–18704.
19. Coon MJ (2005) Cytochrome P450: Nature’s most versatile biological catalyst. Annu
Rev Pharmacol Toxicol 45:1–25.
Washington, DC).
5. Shen XH, Zhou NY, Liu SJ (2012) Degradation and assimilation of aromatic compounds
by Corynebacterium glutamicum: Another potential for applications for this bacte-
rium? Appl Microbiol Biotechnol 95:77–89.
6. Briasoulis A, Palla M, Afonso L (2015) Meta-analysis of the effects of carvedilol versus
metoprolol on all-cause mortality and hospitalizations in patients with heart failure.
Am J Cardiol 115:1111–1115.
20. Behrendorff JB, Huang W, Gillam EM (2015) Directed evolution of cytochrome
P450 enzymes for biocatalysis: Exploiting the catalytic versatility of enzymes with
relaxed substrate specificity. Biochem J 467:1–15.
7. Kesavan L, et al. (2011) Solvent-free oxidation of primary carbon-hydrogen bonds in
toluene using Au-Pd alloy nanoparticles. Science 331:195–199.
8. Newhouse T, Baran PS (2011) If C-H bonds could talk: Selective C-H bond oxidation.
Angew Chem Int Ed Engl 50:3362–3374.
9. Zhang FL, Hong K, Li TJ, Park H, Yu JQ (2016) Organic chemistry. Functionalization of
C(sp3)-H bonds using a transient directing group. Science 351:252–256.
10. Chen MS, White MC (2010) Combined effects on selectivity in Fe-catalyzed methylene
oxidation. Science 327:566–571.
11. Desai LV, Hull KL, Sanford MS (2004) Palladium-catalyzed oxygenation of unactivated
sp3 C-H bonds. J Am Chem Soc 126:9542–9543.
12. Que L, Jr, Tolman WB (2008) Biologically inspired oxidation catalysis. Nature 455:
333–340.
21. McIntosh JA, Farwell CC, Arnold FH (2014) Expanding P450 catalytic reaction space
through evolution and engineering. Curr Opin Chem Biol 19:126–134.
22. Fasan R (2012) Tuning P450 enzymes as oxidation catalysts. ACS Catal 2:647–666.
23. Polic V, Auclair K (2014) Controlling substrate specificity and product regio- and
stereo-selectivities of P450 enzymes without mutagenesis. Bioorg Med Chem 22:
5547–5554.
24. Larsen AT, May EM, Auclair
K (2011) Predictable stereoselective and chemo-
selective hydroxylations and epoxidations with P450 3A4. J Am Chem Soc 133:
7853–7858.
25. Li S, Ouellet H, Sherman DH, Podust LM (2009) Analysis of transient and catalytic
desosamine-binding pockets in cytochrome P-450 PikC from Streptomyces ven-
ezuelae. J Biol Chem 284:5723–5730.
13. Das S, Incarvito CD, Crabtree RH, Brudvig GW (2006) Molecular recognition in the
selective oxygenation of saturated C-H bonds by a dimanganese catalyst. Science 312:
1941–1943.
26. Li S, Podust LM, Sherman DH (2007) Engineering and analysis of a self-sufficient
biosynthetic cytochrome P450 PikC fused to the RhFRED reductase domain. J Am
Chem Soc 129:12940–12941.
14. Das S, Brudvig GW, Crabtree RH (2008) High turnover remote catalytic oxygenation of
alkyl groups: How steric exclusion of unbound substrate contributes to high molec-
ular recognition selectivity. J Am Chem Soc 130:1628–1637.
15. Roiban GD, Agudo R, Reetz MT (2014) Cytochrome P450 catalyzed oxidative hy-
droxylation of achiral organic compounds with simultaneous creation of two chirality
centers in a single C-H activation step. Angew Chem Int Ed Engl 53:8659–8663.
27. Li S, et al. (2009) Selective oxidation of carbolide C-H bonds by an engineered mac-
rolide P450 mono-oxygenase. Proc Natl Acad Sci USA 106:18463–18468.
28. Negretti S, et al. (2014) Directing group-controlled regioselectivity in an enzymatic
C-H bond oxygenation. J Am Chem Soc 136:4901–4904.
29. Narayan AR, et al. (2015) Enzymatic hydroxylation of an unactivated methylene C-H
bond guided by molecular dynamics simulations. Nat Chem 7:653–660.
8 of 9
|
Du et al.