Structural determinants of reductive terpene cyclization in iridoid biosynthesis

Abstract

The carbon skeleton of ecologically and pharmacologically important iridoid monoterpenes is formed in a reductive cyclization reaction unrelated to canonical terpene cyclization. Here we report the crystal structure of the recently discovered iridoid cyclase (from Catharanthus roseus) bound to a mechanism-inspired inhibitor that illuminates substrate binding and catalytic function of the enzyme. Key features that distinguish iridoid synthase from its close homolog progesterone 5β-reductase are highlighted.

Figure 1. Mechanism of iridoid synthase (ISY)

(a). Conversion of 8-oxogeranial into iridodial and nepetalactol. Both products are observed in the ISY reaction. (b). Reduction of progesterone, catalyzed by the close ISY homologue progesterone 5β-reductase (P5βR). (c). Possible mechanistic scenarios for ISY. I) A Rauhut-Currier cyclization covalently assisted by cysteine (or histidine) is followed by reduction. II) Reduction followed by a hetero Diels-Alder reaction. III) Reduction followed by a Michael cyclization. IV) Reduction followed by an imine-assisted Michael cyclization.

Figure 2. Structure of ISY (Catharanthus roseus, CrISY)

The protein backbone is shown in gray, binding pocket residues and the backbone of residue Ile145 as sticks (carbon: green, oxygen: red, nitrogen: blue), NADP⁺ as sticks with carbons in cyan, and additional ligands as sticks in magenta. The side chain of residue Ile145 is omitted for clarity. Relevant polar contacts with bonding distances between 2.5 and 3.5 Å are highlighted with yellow dashes. (a). CrISY co-crystallized with NADP⁺ shows an adventitious ligand in the active site (PDB code 5DCU), most likely triethylene glycol carboxylic acid (TEG). (b). Active site of CrISY co-crystallized with geranic acid (GEA; PDB code 5DF1). The carbons equivalent to the start and end point of the hydride transfer from NADPH to 8-oxogeranial are connected with a black dotted line (distance: 3.4 Å). (c). The enolate form of iridodial (IEN) modeled into the active site of the ‘open conformation’ G150A mutant (see text and Fig. 3).

Figure 3. Open and closed forms of loop Gly150-Val158 in CrISY

All electron density maps are of the form 2mF_obs – dF_calc, contoured at 0.8 σ. (a). TEG bound structure at 1.4 Å resolution revealed the Gly150-Val158 loop in ‘open’ and ‘closed’ conformations. In the closed conformation, Phe152 contacts the ligand. (b). Superposition of the GEA bound structure (gray) and the loop of CrISY^G150A (PDB code 5DCY, green). With GEA bound, this loop is exclusively in the closed conformation and in mutant CrISY^G150A, the loop is exclusively in an open conformation (Supplementary Fig. 14).