3
0
succinate. As discussed above, the presence of a-ketoglutarate
as well as ascorbate was essential for the enzymatic conversion
of fumitremorgin B to verruculogen by FtmOx1 (Fig. 2), which
excludes the possibility that formation of verruculogen and
oxidative decarboxylation of a-ketoglutarate to succinate would
be two independent activities of FtmOx1. Therefore, we postulate
a reaction mechanism of verruculogen formation including Fe(II),
Acknowledgements
This work was supported by the Deutsche Forschungsgesellschaft
and the Deutscher Akadenmischer Austauschdienst (PPP pro-
gram) (to S.-M. Li). We thank Prof. Dr. Rolf M u¨ ller (Saarbr u¨ cken)
for LC-MS analysis.
a-ketoglutarate and ascorbate as well as two O
2
molecules
References
(
Scheme 2).
1
2
J. C. Frisvad, Arch. Environ. Contam. Toxicol., 1989, 18, 452–467.
K. Khoufache, O. Puel, N. Loiseau, M. Delaforge, D. Rivollet, A.
Coste, C. Cordonnier, E. Escudier, F. Botterel and S. Bretagne, BMC
Microbiol., 2007, 7, 5.
According to this hypothesis, formation of the Fe(IV)=O species
E) from Fe(II) (A) via Fe(III) (C) and a trioxo Fe(IV)-complex
D) is in analogy to other known a-ketoglutarate-dependent
(
(
3
4
5
6
R. M. Williams, E. M. Stocking and J. F. Sanz-Cervera, Top. Curr.
Chem., 2000, 209, 97–173.
J. B. Day and P. G. Mantle, Appl. Environ. Microbiol., 1982, 43, 514–
516.
20,30,31
dioxygenases.
The Fe(IV)=O species (E) could then generate a
radical at the a-position to the C=C bond of the N-prenyl moiety
of fumitremorgin B by its own reduction to an Fe(III)–OH species
R. M. Horak and R. Vleggaar, J. Chem. Soc., Chem. Commun., 1987,
(
F) as proposed for TauD from E. coli, where Fe(IV)=O attacks
1
568–1570.
20
the substrate by hydrogen abstraction. This is also plausible,
H. W. Schroeder, R. J. Cole, H. Hein, Jr. and J. W. Kirksey, Appl.
Microbiol., 1975, 29, 857–858.
if the sequence homologues of FtmOx1 from the PhyH family,
18
7 R. Vleggaar, R. M. Horak and V. J. Maharaj, J. Chem. Soc., Chem.
Commun., 1993, 274–275.
which hydroxylate the a-position of a C=O bond, are taken into
consideration. In addition, radical formation in an allylic position
8
J. Willingale, K. P. Perera and P. G. Mantle, Biochem. J., 1983, 214,
91–993.
10
has also been proposed for COX-catalysed peroxide formation.
9
10
Similar to that proposed for COX,
the molecular oxygen
9 P. S. Covello, Phytochemistry, 2008, 69, 2881–2885.
0 L. J. Marnett, Curr. Opin. Chem. Biol., 2000, 4, 545–552.
1 A. Grundmann and S.-M. Li, Microbiology, 2005, 151, 2199–2207.
2 N. Steffan, A. Grundmann, W.-B. Yin, A. Kremer and S.-M. Li, Curr.
Med. Chem., 2009, 16, 218–231.
1
1
1
would then attack the radical at the N-prenyl moiety, resulting
in formation of the radical G, which would undergo cyclization
to enzyme-bound verruculogen radical (H). To complete the
reaction, the verruculogen radical (H) has to abstract a hydrogen
atom to form free or enzyme-bound verruculogen. This could take
place, as indicated by the dashed line in Scheme 2, by abstraction
of one hydrogen atom from the Fe(III)–OH species in H with
involvement of a new molecule of fumitremorgin B and formation
of a Fe(IV)=O species E, which would then undergo another
13 S. Maiya, A. Grundmann, S.-M. Li and G. Turner, ChemBioChem,
2
006, 7, 1062–1069.
1
4 A. Grundmann, T. Kuznetsova, S. S. Afiyatullov and S.-M. Li,
ChemBioChem, 2008, 9, 2059–2063.
15 N. Kato, H. Suzuki, H. Takagi, Y. Asami, H. Kakeya, M. Uramoto, T.
Usui, S. Takahashi, Y. Sugimoto and H. Osada, ChemBioChem, 2009,
1
0, 920–928.
1
6 Y. Ding, R. S. Bojja and L. C. Du, Appl. Environ. Microbiol., 2004, 70,
reaction cycle. However, this hypothesis is in contrast to our
finding that the enzymatic activity was strictly dependent on the
presence of ascorbate. Therefore, reduction of H to enzyme-bound
verruculogen and Fe(II) complex (I) would be more plausible. The
reducing agent/system in vivo for this reaction is as yet unknown,
which could explain the low conversion rate of fumitremorgin B to
verruculogen by FtmOx1 in vitro by using ascorbate as a reducing
agent.
1
931–1934.
17 R. H. Proctor, D. W. Brown, R. D. Plattner and A. E. Desjardins,
Fungal Genet. Biol., 2003, 38, 237–249.
1
8 C. J. Schofield and M. A. McDonough, Biochem. Soc. Trans., 2007, 35,
8
70–875.
19 C. J. Schofield and Z. H. Zhang, Curr. Opin. Struct. Biol., 1999, 9,
22–731.
0 E. G. Kovaleva and J. D. Lipscomb, Nat. Chem. Biol., 2008, 4, 186–
93.
1 T. Rundberget and A. L. Wilkins, J. Chromatogr., A, 2002, 964, 189–
97.
7
2
2
2
2
2
2
1
1
2 E. Eichhorn, J. R. vanderPloeg, M. A. Kertesz and T. Leisinger, J. Biol.
Chem., 1997, 272, 23031–23036.
3 T. A. M u¨ ller, T. Fleischmann, J. R. van der Meer and H. P. E. Kohler,
Appl. Environ. Microbiol., 2006, 72, 4853–4861.
4 G. A. Jansen, S. J. Mihalik, P. A. Watkins, C. Jakobs, H. W. Moser and
R. J. A. Wanders, Clin. Chim. Acta, 1998, 271, 203–211.
Conclusions
The results reported in this study have provided evidence for
the biosynthetic relationship of fumitremorgins to verruculogen.
Furthermore, this study has described the first a-ketoglutarate-
dependent dioxygenase, which functions as an endoperoxide-
forming enzyme. Both oxygen atoms of the endoperoxide bond
5 A. Cultrone, C. Scazzocchio, M. Rochet, G. Montero-Moran, C. Drevet
and R. Fernandez-Martin, Mol. Microbiol., 2005, 57, 276–290.
6 A. G. Prescott and M. D. Lloyd, Nat. Prod. Rep., 2000, 17, 367–383.
7 X. F. Xiao, S. Wolfe and A. L. Demain, Biochem. J., 1991, 280, 471–
2
2
are derived from a single molecule of oxygen (O ), which was
2
4
74.
28 T. Hashimoto, J. Matsuda and Y. Yamada, FEBS Lett., 1993, 329,
5–39.
1
8
clearly demonstrated by incubation of FtmOx1 in an O -enriched
2
3
atmosphere in the presence of fumitremorgin B, Fe(II), ascorbate
and a-ketoglutarate. Further investigations with different reducing
partners such as ferredoxin might give new insights into this novel
enzyme group.
2
3
3
9 E. De Carolis and L. De V, Phytochemistry, 1994, 36, 1093–1107.
0 R. P. Hausinger, Crit. Rev. Biochem. Mol. Biol., 2004, 39, 21–68.
1 J. C. Price, E. W. Barr, L. M. Hoffart, C. Krebs and J. M. Bollinger,
Biochemistry, 2005, 44, 8138–8147.
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