10.1002/cctc.201900606
ChemCatChem
FULL PAPER
Enzyme assays and HPLC analysis
Acknowledgements
A typical reaction mixture was composed of 0.1 mg/mL purified enzyme,
5 mM DTT, 1 mM substrate, 1 mM SAM and 25% E. coli cell lysate, in 50
mM phosphate buffer, pH 7.5. Negative controls were also performed
without adding MTs. Assays were performed in triplicate. Reactions were
incubated at 28°C with 1000 rpm agitation in an Eppendorf Thermomixer.
Samples were taken after 2, 4 and 24 h reaction time and an equal
volume of acetonitrile was added to quench the reactions. The samples
were centrifuged at full speed for 30 min to remove protein precipitate
and then 200 µL supernatant were transferred to HPLC sample vial
inserts prior to HPLC analysis. Analysis were performed on VWR Hitachi
Elite LaChrom system equipped with the Kinetex EVO C18 (4.6 x 250
mm column, 5 µm particle size, Phenomenex) reversed-phase column.
The mobile phase for the separation of isoeugenol (1b) and the
methylated product was 10 mM sodium 1-heptanesulfonate, 20 mM
phosphate, pH 4.4 and 50 % (v/v) acetonitrile; 50% water and methanol
were used for detection of eugenol (2b) and methyl eugenol; 0.1 % acetic
acid and acetonitrile were used for the separation of caffeic acid (3a) (18
% acetonitrile), 3,4-dihydroxybenzoic acid (5a) (10 % acetonitrile), 3,4-
Q.T. would like to thank the China Scholarship Council for
financial support of her PhD thesis project (File No.:
201606150073).
Keywords: biocatalysis • methyltransferases • catalytic
promiscuity • regioselectivity
[1]
[2]
a) N. Koirala, N. H. Thuan, G. P. Ghimire, D. V. Thang, J. K.
Sohng, Enzyme Microb. Technol. 2016, 86, 103-116; b) S. A.
Heleno, A. Martins, M. J. R. Queiroz, I. C. Ferreira, Food Chem.
2015, 173, 501-513.
a) C. D. Davis, E. O. Uthus, Exp. Biol. Med. 2004, 229, 988-995; b)
A. E. McBride, P. A. Silver, Cell 2001, 106, 5-8; c) P. T. Männistö,
S. Kaakkola, Pharmacol. Rev. 1999, 51, 593-628.
[3]
[4]
A. W. Struck, M. L. Thompson, L. S. Wong, J. Micklefield,
ChemBioChem 2012, 13, 2642-2655.
a) S. Mordhorst, J. Siegrist, M. Müller, M. Richter, J. N. Andexer,
Angew. Chem., Int. Ed. 2017, 56, 4037-4041; Angew. Chem. 2017,
129, 4095-4099; b) J. C. Sadler, L. D. Humphreys, R. Snajdrova,
G. A. Burley, ChemBioChem 2017, 18, 992-995; c) J. E.
Farnberger, N. Richter, K. Hiebler, S. Bierbaumer, M. Pickl, W.
Skibar, F. Zepeck, W. Kroutil, Comm. Chem., 2018, 1, Art. No. 82;
d) N. Richter, J. Farnberger, S. Pompei, C. Grimm, W. Skibar, F.
Zepeck, W. Kroutil, Adv. Synth. Catal., 2019, doi:
10.1002/adsc.201801590.
dihydroxyphenylacetic acid (6a) (20
dihydroxyphenylpropanoic acid (7a) (20
%
%
acetonitrile) and 3,4-
acetonitrile) and their
methylated products. A modified linear gradient with 0-2 min 5%-20%
methanol, 2-11 min 23% methanol, 11-13 min 95% methanol, 13-16 min
5% methanol and lasted until 20 min, mixed with 0.1 % acetic acid was
applied for the separations of 3,4-dihydroxybenzaldehyde (4a) and
products.[5d] Wavelengths for the detections of isoeugenol, eugenol,
caffeic acid, 3,4-dihydroxybenzaldehyde and the phenolic acids were
260, 280, 320, 320 and 280 nm, respectively. All analyses were
performed at a flow rate 1 mL/min and the column temperature was
35ºC. The identities of meta-, para- and double-methylated products
were confirmed by comparison with chromatographic elution times of
commercial standards. Substrate and product concentrations were
determined by comparing the peak areas to the calibration curves of
each compound and the conversions were calculated. The kinetic
[5]
a) S. Singh, J. Zhang, T. D. Huber, M. Sunkara, K. Hurley, R. D.
Goff, G. Wang, W. Zhang, C. Liu, J. Rohr, Angew. Chem., Int. Ed.
2014, 53, 3965-3969; Angew. Chem., 2014, 126, 4046-4050; b) B.
W. K. Lee, H. G. Sun, T. Zang, B. J. Kim, J. F. Alfaro, Z. S. Zhou, J.
Am. Chem. Soc. 2010, 132, 3642-3643; c) M. Tengg, H. Stecher, L.
Offner, K. Plasch, F. Anderl, H. Weber, H. Schwab, M. Gruber-
Khadjawi, ChemCatChem 2016, 8, 1354-1360; d) B. J. C. Law, M.
R. Bennett, M. L. Thompson, C. Levy, S. A. Shepherd, D. Leys, J.
Micklefield, Angew. Chem. Int. Ed. 2016, 55, 2683-2687; Angew.
Chem. 2016, 128, 2733-2737.
constants (KM,obs) of IeOMT_T133M were determined at various
substrate concentrations and 1 mM SAM under the same reaction
condition as in the enzyme assays using appropriate amounts of purified
enzyme. Initial reaction rates were measured and fit to the Michaelis-
Menten model using GraphPad Prism 6.0 (GraphPad Software Inc.) to
determined the Vmax, obs and KM,obs values for each substrate. Then kcat,obs
[6]
[7]
I. J. Kopin, Pharmacol. Rev. 1985, 37, 333-364.
C. R. Creveling, N. Dalgard, H. Shimizu, J. W. Daly, Mol.
Pharmacol. 1970, 6, 691-696.
was defined by dividing Vmax,obs by the enzyme concentration and kcat,obs
KM,obs was calculated.
/
[8]
[9]
N. J. Walton, A. Narbad, C. Faulds, G. Williamson, Curr. Opin.
Biotechnol. 2000, 11, 490-496.
a) J. T. Nelson, J. Lee, J. W. Sims, E. W. Schmidt, Appl. Environ.
Microbiol. 2007, 73, 3575-3580; b) J. Siegrist, J. Netzer, S.
Mordhorst, L. Karst, S. Gerhardt, O. Einsle, M. Richter, J. N.
Andexer, FEBS Lett. 2017, 591, 312-321.
Bioinformatic analysis
The bioinformatic analysis was performed with YASARA 18.11.21. First
the structure of 3REO was back-mutated to its wild-type sequence and
the SAH was transformed to SAM by the addition of the methyl group
and this structure was refined at pH 7.5, 25ºC for 500ps, taking a
snapshot every 25 ps. The structure with the lowest energy was selected
for the further experiments. For the mutants, the respective amino acids
were swapped with subsequent energy minimization. The same was
performed for the exchange of substrate..As we knew the binding pattern
of isoeugenol, we swapped the atoms of the R-group to form the caffeic
acid and subsequently minimized the energy. The figures were prepared
with Pymol. The RIN analysis was performed with RINalyzer according to
literature, with the use of Cytoscape 3.7 and Chimera 1.13.[23]
[10]
[11]
D. Guo, F. Chen, K. Inoue, J. W. Blount, R. A. Dixon, The Plant
Cell 2001, 13, 73-88.
a) J. Ralph, K. Lundquist, G. Brunow, F. Lu, H. Kim, P. F. Schatz, J.
M. Marita, R. D. Hatfield, S. A. Ralph, J. H. Christensen,
Phytochem. Rev. 2004, 3, 29-60; b) L. B. Davin, N. G. Lewis, Curr.
Opin. Biotechnol. 2005, 16, 398-406.
[12]
[13]
[14]
P. M. Dewick, Medicinal natural products: a biosynthetic approach,
John Wiley & Sons, 2002.
J. Wang, E. Pichersky, Arch. Biochem. Biophys. 1998, 349, 153-
160.
J. Wang, E. Pichersky, Arch. Biochem. Biophys. 1999, 368, 172-
180.
[15]
[16]
G. V. Louie, M. E. Bowman, Y. Tu, A. Mouradov, G. Spangenberg,
J. P. Noel, The Plant Cell 2010, 22, 4114-4127.
[17]
C. Zubieta, X.-Z. He, R. A. Dixon, J. P. Noel, Nat. Struct. Mol. Biol.
2001, 8, 271.
This article is protected by copyright. All rights reserved.