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ChemComm
DOI: 10.1039/C6CC09213F
COMMUNICATION
ChemComm
The FlA1-5’-ClDA enzyme complex model when compared with
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D. O'Hagan and H. Deng, Chem. Rev. (Washington, DC, U. S.),
015, 115, 634.
C. D. Cadicamo, J. Courtieu, H. Deng, A. Meddour and D.
O'Hagan, ChemBioChem, 2004, 5, 685.
C. Dong, F. Huang, H. Deng, C. Schaffrath, J. B. Spencer, D.
O'Hagan and J. H. Naismith, Nature, 2004, 427, 561.
0 X. Zhu, D. A. Robinson, A. R. McEwan, D. O'Hagan and J. H.
Naismith, J. Am. Chem. Soc., 2007, 129, 14597.
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the bound models for 6 – 11 identifies the main reason for the
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diminished or loss of activity. The R substituent of amino group is
hydrogen-bonded to the enzyme through the amide side-chain of
N215, and the peptide carbonyl of R277 (Figure 2C). The loss of this
group removes this interaction, which would lower the affinity of
the enzyme for the substrate, and substituting with chlorine (6 and
7
), hydrogen (8 and 9) or oxygen (11) will not form any stabilizing
1 H. Deng, S. L. Cobb, A. R. McEwan, R. P. McGlinchey, J. H.
Naismith, D. O'Hagan, D. A. Robinson and J. B. Spencer, Angew.
Chem. Int. Ed., 2006, 45, 759; Angew. Chem., 2006, 118, 773.
hydrogen bonds with N215 (Figure 2D and Figure S1A-B). In the
case of 10, the substituted methoxy group could be too bulky to fit
into this pocket, and thus cannot form any stabilizing hydrogen 12 S. L. Cobb, H. Deng, A. R. McEwan, J. H. Naismith, D. O'Hagan
bonds (Figure S1C). We tried to mutate N215 in order to explore
potential alterations in specificity at the C-6 position, but so far the
mutations we tested (N215LQ) have rendered the protein insoluble
and D. A. Robinson, Org. Biomol. Chem., 2006, 4, 1458.
3 M. Thomsen, S. B. Vogensen, J. Buchardt, M. D. Burkart and R.
P. Clausen, Org. Biomol. Chem., 2013, 11, 7606.
4 M. E. Sergeev, F. Morgia, M. R. Javed, M. Doi and P. Y. Keng, J.
Mol. Catal. B: Enzym., 2013, 97, 74.
5 S. Thompson, Q. Zhang, M. Onega, S. McMahon, I. Fleming, S.
Ashworth, J. H. Naismith, J. Passchier and D. O'Hagan, Angew.
Chem. Int. Ed., 2014, 53, 8913; Angew. Chem., 2014, 126, 9059.
6 S. Thompson, M. Onega, S. Ashworth, I. N. Fleming, J. Passchier
and D. O'Hagan, Chem. Commun. (Cambridge, U. K.), 2015, 51,
13542.
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(data not shown). The addition of an amino group at the C-2
position helps to compensate the loss of key interactions at C-6
position and improve the binding of some substrates (e.g. 6 and 8)
due to the additional hydrogen bonding (vide supra), as well as the
favourable van der Waals interactions from A279L and Y.
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In conclusion, we found that substitutions at the C-2 position of
the adenine group are sensitive to steric bulk whereas an amino
group at the C-2 position led to enhanced fluorination yield. 17 S. Thompson, I. N. Fleming and D. O'Hagan, Org. Biomol. Chem.,
Additionally, our evolved FlA1 fluorinase variants are active against
substrates that are modified at the C-2 and/or C-6 positions of the
adenine ring despite being evolved against 5’-ClDA 1 and ʟ-Met
substrates. Modifications at F213 and/or A279 residues showed
improved activity over FlA1 on the eight new substrates tested
including novel activity for two of the substrates, 4 and 7. Based on
2016, 14, 3120.
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8 H. Sun, W. L. Yeo, Y. H. Lim, X. Chew, D. J. Smith, B. Xue, K. P.
Chan, R. C. Robinson, E. G. Robins, H. Zhao and E. L. Ang,
Angew. Chem. Int. Ed., 2016, 55, 14277; Angew. Chem., 2016,
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28, 14489.
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9 H. Deng, S. L. Cobb, A. D. Gee, A. Lockhart, L. Martarello, R. P.
McGlinchey, D. O'Hagan and M. Onega, Chem. Commun.
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our previous kinetic studies, it is likely that the improvement in
activity shown by our enzyme variants over FlA1 for these
substrates is due to the enhancement of the first step, i.e.
conversion of the substrate and ʟ-Met to a SAM-analog in this two-
(
Cambridge, U. K.), 2006, 652.
0 M. Onega, J. Domarkas, H. Deng, L. F. Schweiger, T. A. Smith, A.
E. Welch, C. Plisson, A. D. Gee and D. O'Hagan, Chem. Commun.
(Cambridge, U. K.), 2010, 46, 139.
step fluorination process. This work has shed light on the specificity 21 M. Winkler, J. Domarkas, L. F. Schweiger and D. O'Hagan,
of the FlA1 fluorinase with respect to modifications at the adenine
moiety of the 5’-ClDA substrate and the mutations that can improve
the promiscuity of the enzyme against non-native substrates. We
believe that our work can serve as starting points for engineering of
the enzyme to perform fluorination on more structurally diverse
small molecules.
Angew. Chem. Int. Ed., 2008, 47, 10141; Angew. Chem., 2008,
20, 10295.
2 S. D. Mills, A. E. Eakin, E. T. Buurman, J. V. Newman, N. Gao, H.
Huynh, K. D. Johnson, S. Lahiri, A. B. Shapiro, G. K. Walkup, W.
Yang and S. S. Stokes, Antimicrob. Agents Chemother., 2011, 55,
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088.
3 S. S. Stokes, H. Huynh, M. Gowravaram, R. Albert, M. Cavero-
Tomas, B. Chen, J. Harang, J. T. Loch, 3rd, M. Lu, G. B. Mullen, S.
Zhao, C. F. Liu and S. D. Mills, Bioorg. Med. Chem. Lett., 2011,
21, 4556.
This work was funded by GlaxoSmithKline – Singapore Economic
Development Board Partnership for Green and Sustainable
Manufacturing and A*STAR ICES. We would like to thank the
members of MERL for their suggestions and comments and Ms 24 S. S. Stokes, M. Gowravaram, H. Huynh, M. Lu, G. B. Mullen, B.
Doris Tan (ICES) for HRMS analyses.
Chen, R. Albert, T. J. O'Shea, M. T. Rooney, H. Hu, J. V. Newman
and S. D. Mills, Bioorg. Med. Chem. Lett., 2012, 22, 85.
Notes and references
25 Y. Song, F. DiMaio, R. Y. Wang, D. Kim, C. Miles, T. Brunette, J.
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