Organic Letters
Letter
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flavin hydroperoxide FADH−OOH, which is attacked by the
aromatic nitrogen of 6, resulting in release of FADH−OH and 2.
A dehydration of FADH−OH regenerates the oxidized FAD.
In the enzyme assays, LaPhzNO1 was unable to convert 5 to
any of the N-oxide products (Figure 5). Moreover, LaPhzNO1
could only convert 6 into 2 but not 1. These results suggest that
the N-oxidation steps must take place before the adjacent O-
methylation steps. Once the hydroxy groups at the C1 and C6
positions are methylated, LaPhzNO1 is no longer able to oxidize
the nitrogen atom. Using LaPhzS and LaPhNO1 coupled
reactions, we confirmed the substrate selectivity of LaPhzNO1.
To further prove this point, we tested a group of N-containing
aromatic compounds as potential substrates, including 8-
hydroxyquinoline (8-HQ), 6-hydroxyquinoline (6-HQ), quino-
line, quinoxaline, quinine, and 2-phenylpyridine (Figure S13).
LaPhzNO1 was able to convert 8-HQ into its oxide in the
presence of NADPH but unable to use any of the other
compounds as substrates. This result not only confirms the
importance of the β-hydroxy for the LaPhzNO1-catalyzed N-
oxdidation but also shows a potential use of this enzyme in the
chemoenzymatic synthesis of aromatic N-oxides, as there are
over 6000 synthetic phenazine compounds and over 150 natural
phenazines.2 Further investigation is needed to elucidate the
molecular basis of the observed substrate selectivity. In
conclusion, LaPhzNO1 represents the first experimentally
characterized aromatic N-monooxygenase. This study may
access to other aromatic N-oxide natural products that possess
fascinating chemical structures and potent biological activities.
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R.; Hiller, W.; Costisella, B.; Thomashow, L. S.; Mavrodi, D. V.;
Blankenfeldt, W. J. Am. Chem. Soc. 2008, 130, 17053.
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge on the
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S
(13) (a) Mavrodi, D. V.; Bonsall, R. F.; Delaney, S. M.; Soule, M. J.;
Phillips, G.; Thomashow, L. S. J. Bacteriol. 2001, 183, 6454.
(b) Greenhagen, B. T.; Shi, K.; Robinson, H.; Gamage, S.; Bera, A.
K.; Ladner, J. E.; Parsons, J. F. Biochemistry 2008, 47, 5281.
(14) Parsons, J. F.; Greenhagen, B. T.; Shi, K.; Calabrese, K.; Robinson,
H.; Ladner, J. E. Biochemistry 2007, 46, 1821.
(15) (a) Chen, Y. C. J.; Peoples, O. P.; Walsh, C. T. J. Bacteriol. 1988,
170, 781. (b) Iwaki, H.; Hasegawa, Y.; Teraoka, M.; Tokuyama, T.;
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Details of experimental procedures, construction of gene
inactivation plasmids and verification of mutants, protein
expression in E. coli and enzyme purification, reactions and
activity assay, and spectroscopic data (PDF)
AUTHOR INFORMATION
Corresponding Authors
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Author Contributions
∥Y.Z. and G.Q. contributed equally.
Notes
̀
(c) Brzostowicz, P. C.; Blasko, M. S.; Rouviere, P. E. Appl. Microbiol.
Biotechnol. 2002, 58, 781. (d) van Berkel, W. J.; Kamerbeek, N. M.;
Fraaije, M. W. J. J. Biotechnol. 2006, 124, 670. (e) Mirza, I. A.; Yachnin, B.
J.; Wang, S.; Grosse, S.; Bergeron, H.; Imura, A.; Iwaki, H.; Hasegawa, Y.;
Lau, P. C. K.; Berghuis, A. M. J. Am. Chem. Soc. 2009, 131, 8848.
(16) (a) Kendrew, S. G.; Hopwood, D. A.; Marsh, E. N. G. J. Bacteriol.
1997, 179, 4305. (b) Shen, B.; Hutchinson, C. R. Biochemistry 1993, 32,
6656.
The authors declare no competing financial interest.
(17) Price-Whelan, A.; Dietrich, L. E. P.; Newman, D. K. Nat. Chem.
Biol. 2006, 2, 71.
(18) Zeyhle, P.; Bauer, J. S.; Kalinowski, J.; Shin-ya, K.; Gross, H.;
Heide, L. PLoS One 2014, 9, e99122.
ACKNOWLEDGMENTS
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This work was supported in part by the NIH (R01AI097260),
NSFC (31329005, 31371981, and 31572046), National Basic
Research (973) program of China (2015CB150600), and Jiangsu
Provincal Key Technology Support Program (BE2015354). Y.Z.
was partially supported by China Scholarship Council. We thank
Dr. Xiuli Wu for assistance in NMR data analysis, and Dr. Ronald
Cerny and Kurt Wulser for assistance in LC-MS and GC-MS
experiments.
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