Crystallographic trapping in the rebeccamycin biosynthetic enzyme RebC

Putative monooxygenase, Putative FAD-monooxygenase

No reasonable conformation for residues 417-425 could be built, despite the presence of electron density for this loop. Related structures 2R0C and 2R0P also show disorder in this region, and the loop is likely to occupy multiple, overlapping orientations in this structure.

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 1 mM K252c for 11 days. 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 1 mM K252c and then flash-frozen in liquid nitrogen., VAPOR DIFFUSION, HANGING DROP, temperature 298K

Structure of the native form of the rebeccamycin biosynthetic enzyme RebC

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

flavin adenine dinucleotide, K252c, Monooxygenase, OXIDOREDUCTASE