Structural basis for the promiscuous biosynthetic prenylation of aromatic natural products

Abstract

The anti-oxidant naphterpin is a natural product containing a polyketide-based aromatic core with an attached 10-carbon geranyl group derived from isoprenoid (terpene) metabolism. Hybrid natural products such as naphterpin that contain 5-carbon (dimethylallyl), 10-carbon (geranyl) or 15-carbon (farnesyl) isoprenoid chains possess biological activities distinct from their non-prenylated aromatic precursors. These hybrid natural products represent new anti-microbial, anti-oxidant, anti-inflammatory, anti-viral and anti-cancer compounds. A small number of aromatic prenyltransferases (PTases) responsible for prenyl group attachment have only recently been isolated and characterized. Here we report the gene identification, biochemical characterization and high-resolution X-ray crystal structures of an architecturally novel aromatic PTase, Orf2 from Streptomyces sp. strain CL190, with substrates and substrate analogues bound. In vivo, Orf2 attaches a geranyl group to a 1,3,6,8-tetrahydroxynaphthalene-derived polyketide during naphterpin biosynthesis. In vitro, Orf2 catalyses carbon-carbon-based and carbon-oxygen-based prenylation of a diverse collection of hydroxyl-containing aromatic acceptors of synthetic, microbial and plant origin. These crystal structures, coupled with in vitro assays, provide a basis for understanding and potentially manipulating the regio-specific prenylation of aromatic small molecules using this structurally unique family of aromatic PTases.

Figure 1. Biosynthesis of naphterpin and other hybrid isoprenoid-polyketide compounds produced by Actinomycetes

The synthesis of naphterpin involves the prenylation of THN, flaviolin or a derived metabolite using a GPP co-substrate. The THN skeleton is further modified, prenylated and incorporated into hybrid isoprenoid-polyketides such as naphterpin, furaquinocin A, napyradiomycin A and marinone. Each isoprenoid C5 unit is shown in red.

Figure 2. Enzymatic assays of Orf2

a, Mg²⁺-dependent prenylation of 1,6-DHN. In lane 1 (control), Orf2 was boiled before addition. The reaction mixture analysed in lane 2 contained no MgCl₂, whereas 5 mM MgCl₂ was added in lane 3. b, Promiscuous activity against chemically diverse aromatic acceptors. Assays used the substrates named and numbered on the left side of the TLC. The chemical structures of four reaction products were determined by MS/NMR analyses and are shown to the right of the TLC.

Figure 3. Structures of Orf2’s PT barrel

a, Stereo view of the Orf2 monomer viewed with the central barrel axis oriented vertically in the plane of the page. The cylindrical solvent-filled β-sheet and outer belt of α-helices are differentially coloured. Bound GSPP and 1,6-DHN reside within the PT barrel and are represented as colour-coded sticks. Mg²⁺ is shown as a gold-coloured van der Waal’s sphere. b, Orf2 substrate/substrate analogue complexes. Only the β-strands (minus β-strands 1 and 10) plus the C-terminal α-helix are displayed in the same vertical orientation as in a after a 30° anticlockwise rotation around the barrel axis. Hydrogen and coordination bonds are shown as green spheres.

Figure 4. Mg²⁺ dependence and substrate recognition in aromatic PTases

a, Representative 2F_o − F_c electron density map, contoured at 1.0σ, displaying the octahedral coordination of the catalytic Mg²⁺. The orientation is the same as that in Figs 3b and 4b after a 180° rotation around the vertical barrel axis. b, Schematic representation of Orf2’s active site. The side chains involved in Mg²⁺, GSPP and 1,6-DHN binding are depicted with direct hydrogen and coordination bonds shown as green dashes and water-mediated bonds as blue dashes. Half circles depict van der Waal’s contacts with grey for residues in the back of the GSPP–1,6-DHN plane, black in the same plane and thick black in the front. c, Biochemical characterization of HypSc. PTase activity was assayed and visualized as in Fig. 2a using 1,6-DHN as a prenyl acceptor and either DMAPP or GPP as potential prenyl donors.