Crystallographic trapping in the rebeccamycin biosynthetic enzyme RebC.
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.
Ryan
Katherine S
KS
Department of Biology, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139, USA.
Howard-Jones
Annaleise R
AR
Hamill
Michael J
MJ
Elliott
Sean J
SJ
Walsh
Christopher T
CT
Drennan
Catherine L
CL
D000970:Antineoplastic Agents / Q000737:chemistry / Q000494:pharmacology; D001426:Bacterial Proteins / Q000737:chemistry*; D002227:Carbazoles / Q000737:chemistry* / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D018360:Crystallography, X-Ray; D005415:Flavins / Q000737:chemistry; D006094:Gram-Positive Bacteria / Q000201:enzymology; D007211:Indoles / Q000737:chemistry* / Q000378:metabolism; D006899:Mixed Function Oxygenases / Q000235:genetics; D008956:Models, Chemical; D008968:Molecular Conformation; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D011487:Protein Conformation; D013379:Substrate Specificity
D000970:Antineoplastic Agents; D001426:Bacterial Proteins; D002227:Carbazoles; D005415:Flavins; D007211:Indoles; C053339:rebeccamycin; D006899:Mixed Function Oxygenases