Person:

Walsh, Christopher

Previous studies of the biosynthetic enzymes involved in the assembly of scytonemin (1), a cyanobacterial sunscreen, have identified β-ketoacid 2 as an important intermediate that is produced by ThDP-dependent enzyme ScyA. We now report that ScyC, previously annotated as a hypothetical protein, catalyzes cyclization and decarboxylation of 2 to generate ketone 5. Assembly of the cyclopentyl[b]indole structure in this manner has little precedent in the chemical literature. Additional mechanistic experiments have revealed that cyclization likely precedes decarboxylation and that the latter event may provide a driving force for cyclopentane formation.

RebC is a putative flavin hydroxylase functioning together with RebP to carry out a key step in the biosynthesis of rebeccamycin. To probe the mechanism of flavin-based chemistry in RebC, we solved the structure of RebC with reduced flavin. Upon flavin reduction, the RebC crystal undergoes a change in its unit cell dimension concurrent with a 5 Å movement of the isoalloxazine ring, positioning the flavin ring adjacent to the substrate-binding pocket. Additionally, a disordered helix becomes ordered upon flavin reduction, closing off one side of the substrate-binding pocket. This structure, along with previously reported structures, increases our understanding of the RebC enzyme mechanism, indicating that either the reduction of the flavin itself or binding of substrate is sufficient to drive major conformational changes in RebC to generate a closed active site. Our finding that flavin reduction seals the active site such that substrate cannot enter suggests that our reduced flavin RebC structure is off-pathway and that substrate binding is likely to precede flavin reduction during catalysis. Along with kinetic data presented here, these structures suggest that the first cycle of catalysis in RebC may resemble that of p-hydroxybenzoate hydroxylase, with substrate binding promoting flavin reduction.

The cyanobacterial natural product scytonemin (1) functions as a sunscreen, absorbing harmful UV-A radiation. Using information from a recently identified gene cluster, we propose a biosynthetic route to this pigment. We also report the characterization of two enzymes, NpR1275 and NpR1276, which are involved in the initial stages of this pathway. A regioselective acyloin reaction between indole-3-pyruvic acid (4) and p-hydroxyphenylpyruvic acid (5) is a key step in assembling the carbon framework of a proposed monomeric scytonemin precursor (2).