title: An unusual role for a mobile flavin in StaC-like indolocarbazole biosynthetic enzymes.
abstract: The indolocarbazole biosynthetic enzymes StaC, InkE, RebC, and AtmC mediate the degree of oxidation of chromopyrrolic acid on route to the natural products staurosporine, K252a, rebeccamycin, and AT2433-A1, respectively. Here, we show that StaC and InkE, which mediate a net 4-electron oxidation, bind FAD with a micromolar K(d), whereas RebC and AtmC, which mediate a net 8-electron oxidation, bind FAD with a nanomolar K(d) while displaying the same FAD redox properties. We further create RebC-10x, a RebC protein with ten StaC-like amino acid substitutions outside of previously characterized FAD-binding motifs and the complementary StaC-10x. We find that these mutations mediate both FAD affinity and product specificity, with RebC-10x displaying higher StaC activity than StaC itself. X-ray structures of this StaC catalyst identify the substrate of StaC as 7-carboxy-K252c and suggest a unique mechanism for this FAD-dependent enzyme.
authors: Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
authors: Peter J
authors: PJ
authors: Goldman
authors: Katherine S
authors: KS
authors: Ryan
authors: Michael J
authors: MJ
authors: Hamill
authors: Annaleise R
authors: AR
authors: Howard-Jones
authors: Christopher T
authors: CT
authors: Walsh
authors: Sean J
authors: SJ
authors: Elliott
authors: Catherine L
authors: CL
authors: Drennan
mesh_terms: D055162:Biocatalysis; D002227:Carbazoles / Q000737:chemistry / Q000378:metabolism*; D005415:Flavins / Q000378:metabolism*; D006899:Mixed Function Oxygenases / Q000378:metabolism*; D008958:Models, Molecular; D015394:Molecular Structure
chemical_list: D002227:Carbazoles; D005415:Flavins; D006899:Mixed Function Oxygenases