Chromopyrrolic acid-soaked RebC with bound 7-carboxy-K252c

7-HYDROXY-12,13-DIHYDRO-6H-INDOLO[2,3-A]PYRROLO[3,4-C]CARBAZOLE-5-CARBOXYLIC ACID

Crystallographic trapping in the rebeccamycin biosynthetic enzyme RebC

Putative monooxygenase, Putative FAD-monooxygenase

MGSSHHHHHHSSGLVPRGSHMNAPIETDVLILGGGPVGMALALDLAHRQVGHLVVEQTDGTITHPRVGTIGPRSMELFRR
WGVAKQIRTAGWPGDHPLDAAWVTRVGGHEVYRIPLGTADTRATPEHTPEPDAICPQHWLAPLLAEAVGERLRTRSRLDS
FEQRDDHVRATITDLRTGATRAVHARYLVACDGASSPTRKALGIDAPPRHRTQVFRNILFRAPELRSLLGERAALFFFLM
LSSSLRFPLRALDGRGLYRLTVGVDDASKSTMDSFELVRRAVAFDTEIEVLSDSEWHLTHRVADSFSAGRVFLTGDAAHT
LSPSGGFGMNTGIGSAADLGWKLAATLRGWAGPGLLATYEEERRPVAITSLEEANVNLRRTMDRELPPGLHDDGPRGERI
RAAVAEKLERSGARREFDAPGIHFGHTYRSSIVCGEPETEVATGGWRPSARPGARAPHAWLTPTTSTLDLFGRGFVLLSF
GTTDGVEAVTRAFADRHVPLETVTCHAPEIHALYERAHVLVRPDGHVAWRGDHLPAELGGLVDKVRGAA

1.5 microliters of RebC (9 mg/mL in 150 mM NaCl, 10% glycerol, 25 mM HEPES pH 7.5) was incubated with 0.35 microliters of guanidine-HCl for 30 seconds, followed by addition of 1.5 microliters of precipitant solution (19% PEG-8000, 0.1 M HEPES pH 7.4), without mixing, at room temperature and sealed over a precipitant well solution. Immediately after set up, crystal trays were placed on a gel shaker and then, after 12 hours, transferred to a storage space in vibration-isolation. A crystal was then soaked in 19% PEG-8000, 0.1 M HEPES pH 7.4, and 5 mM chromopyrrolic acid for 1 week. The crystal was then soaked for 5 seconds in a cryogenic solution containing 19% PEG-8000, 0.1 M HEPES pH 7.4, 20% glycerol, and 5 mM chromopyrrolic acid and then flash-frozen in liquid nitrogen, VAPOR DIFFUSION, HANGING DROP, temperature 298K

Structure of the substrate-free form of the rebeccamycin biosynthetic enzyme RebC

flavin adenine dinucleotide, chromopyrrolic acid, 7-carboxy-K252c, Monooxygenase, OXIDOREDUCTASE

MNAPIETDVLILGGGPVGMALALDLAHRQVGHLVVEQTDGTITHPRVGTIGPRSMELFRRWGVAKQIRTAGWPGDHPLDA
AWVTRVGGHEVYRIPLGTADTRATPEHTPEPDAICPQHWLAPLLAEAVGERLRTRSRLDSFEQRDDHVRATITDLRTGAT
RAVHARYLVACDGASSPTRKALGIDAPPRHRTQVFRNILFRAPELRSLLGERAALFFFLMLSSSLRFPLRALDGRGLYRL
TVGVDDASKSTMDSFELVRRAVAFDTEIEVLSDSEWHLTHRVADSFSAGRVFLTGDAAHTLSPSGGFGMNTGIGSAADLG
WKLAATLRGWAGPGLLATYEEERRPVAITSLEEANVNLRRTMDRELPPGLHDDGPRGERIRAAVAEKLERSGARREFDAP
GIHFGHTYRSSIVCGEPETEVATGGWRPSARPGARAPHAWLTPTTSTLDLFGRGFVLLSFGTTDGVEAVTRAFADRHVPL
ETVTCHAPEIHALYERAHVLVRPDGHVAWRGDHLPAELGGLVDKVRGAA