Structural studies of rebeccamycin, staurosporine, and violacein biosynthetic enzymes

Abstract

The biosynthesis of medically relevant bisindole natural products rebeccamycin, staurosporine, and violacein from the common starting material L-tryptophan involves shared enzymatic transformations. However, the pathways diverge at two steps, each involving a reactive, bisindole intermediate. We have taken a structural approach to characterize the biosynthetic enzymes responsible for these divergence points in each pathway. One major difference between rebeccamycin and staurosporine is the oxidative state of the C-7 carbon. The enzymes RebC and StaC (65% sequence identity) control the oxidative outcome at the C-7 position. Our work on the rebeccamycin biosynthetic enzyme RebC has enabled us to crystallographically 'trap' its putative substrate and to identify a likely enzymatic function for RebC in controlling the outcome of this key step in rebeccamycin biosynthesis. We have also used the structure of RebC with reduced flavin to probe the likely reaction cycle of a single round of flavin-based hydroxylation chemistry in RebC. Finally, the structure of RebC has allowed us to use a structure-based mutagenesis approach to install a higher affinity binding site for FAD in the RebC homologue StaC, which normally binds FAD weakly. The resulting protein possesses RebC-like properties. Another divergence point between these biosynthetic pathways is the presence or absence of the VioE enzyme, which diverts a reactive intermediate toward violacein precursor. Our structural studies on VioE have shown that this enzyme shares a fold with lipoprotein carrier proteins. A series of site-directed mutagenesis experiments on residues around a PEG molecule bound in the VioE structure have revealed the likely location of the active site in VioE.