An unusual role for a mobile flavin in StaC-like indolocarbazole biosynthetic enzymes.

Publication Type:

Journal Article

Authors:

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

Source:

Chem Biol, Volume 19, Issue 7, p.855-65 (2012)

Keywords:

Biocatalysis, Carbazoles, Flavins, Mixed Function Oxygenases, Models, Molecular, Molecular Structure

Abstract:

<p>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.</p>