Crystallographic trapping in the rebeccamycin biosynthetic enzyme RebC.

Publication Type:

Journal Article

Authors:

Ryan, Katherine S; Howard-Jones, Annaleise R; Hamill, Michael J; Elliott, Sean J; Walsh, Christopher T; Drennan, Catherine L

Source:

Proc Natl Acad Sci U S A, Volume 104, Issue 39, p.15311-6 (2007)

Keywords:

Antineoplastic Agents, Bacterial Proteins, Carbazoles, Chromatography, High Pressure Liquid, Crystallography, X-Ray, Flavins, Gram-Positive Bacteria, Indoles, Mixed Function Oxygenases, Models, Chemical, Molecular Conformation, Nucleic Acid Conformation, Protein Binding, Protein Conformation, Substrate Specificity

Abstract:

<p>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.</p>