Crystallographic trapping in the rebeccamycin biosynthetic enzyme RebC
- Katherine S. Ryan*,
- Annaleise R. Howard-Jones†,
- Michael J. Hamill‡,
- Sean J. Elliott‡,
- Christopher T. Walsh†,§, and
- Catherine L. Drennan*,§,¶
- Departments of *Biology and
- ¶Chemistry, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139;
- †Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115; and
- ‡Department of Chemistry, Boston University, 590 Commonwealth Avenue, Boston, MA 02215
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Contributed by Christopher T. Walsh, July 31, 2007 (received for review July 15, 2007)
Abstract
The biosynthesis of rebeccamycin, an antitumor compound, involves the remarkable eight-electron oxidation of chlorinated chromopyrrolic acid. Although one rebeccamycin biosynthetic enzyme is capable of generating low levels of the eight-electron oxidation product on its own, a second protein, RebC, is required to accelerate product formation and eliminate side reactions. However, the mode of action of RebC was largely unknown. Using crystallography, we have determined a likely function for RebC as a flavin hydroxylase, captured two snapshots of its dynamic catalytic cycle, and trapped a reactive molecule, a putative substrate, in its binding pocket. These studies strongly suggest that the role of RebC is to sequester a reactive intermediate produced by its partner protein and to react with it enzymatically, preventing its conversion to a suite of degradation products that includes, at low levels, the desired product.
Footnotes
- §To whom correspondence may be addressed. E-mail: cdrennan{at}mit.edu or christopher_walsh{at}hms.harvard.edu
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Author contributions: K.S.R., A.R.H.-J., and M.J.H. performed research; A.R.H.-J. and C.T.W. contributed new reagents/analytic tools; K.S.R., A.R.H.-J., M.J.H., S.J.E., C.T.W., and C.L.D. analyzed data; and K.S.R. and C.L.D. wrote the paper.
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The authors declare no conflict of interest.
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Data deposition: The atomic coordinates and structure factors have been deposited in the Protein Data Bank, www.pdb.org (PDB ID codes 2R0C, 2R0G, and 2R0P).
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This article contains supporting information online at www.pnas.org/cgi/content/full/0707190104/DC1.
- © 2007 by The National Academy of Sciences of the USA
