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Structural determinants of reductive terpene cyclization in iridoid biosynthesis

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 (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 homologue, progesterone 5β-reductase, are highlighted.

A full understanding of iridoid cyclization has not emerged. Rauhut-Currier cyclization, in which covalently assisted cyclization occurs before reduction 12 (Fig. 1c) is suggested by the presence of non-reduced iridoids isolated from beetles. 13 However, CrISY produces minor quantities of reduced, non-cyclized products, 6 suggesting that in plants reduction of 8oxogeranial precedes cyclization. We proposed that, consistent with the P5βR mechanism, 14 an enol or enolate intermediate initially forms via a 1,4 reduction of the substrate by NADPH. 6 ISY could then catalyze cyclization of the enol/enolate. One cyclization mechanism uses a hetero Diels-Alder reaction to form nepetalactol (Fig. 1c), though recent studies with substrate analogs argue against this concerted cycloaddition. 15 More likely, the enolate cyclizes via a Michael reaction to form iridodial (Fig. 1c), which is the open dial form of nepetalactol (Fig. 1a). Michael cyclization could be assisted by covalent catalysis, such as imine formation at C8 with an active site lysine (Fig. 1c). 16 To clarify the mechanism, CrISY's structure was solved in its apo form and with NADP + bound (PDB codes 5DCW and 5DCU, respectively) at 1.90 Å and 1.40 Å resolution, respectively (Fig. 2a Table 2). 17 The structure of P5βR from Digitalis lanata (DlP5βR, PDB code 2V6G, 14 Table 3), which has an overall fold that is highly similar to CrISY, was used for molecular replacement. The tyrosine and lysine active site residues that are used in the model for P5βR catalysis, along with many of the residues that define the shape of the P5βR active site, are conserved in CrISY ( Supplementary Fig. 2b). 14 The apo and NADP + bound forms of CrISY exhibit minor differences in conformation ( Supplementary Fig. 2, Supplementary Table 3). 14 An adventitious ligand, most likely an oxidized polyethylene glycol fragment (triethylene glycol carboxylic acid, TEG), was observed in the active site of the NADP + bound enzyme (Fig.  2a, Supplementary Fig. 3a,b). Since efforts to co-crystallize CrISY with substrate, product and/or NADPH were unsuccessful, we searched for an inhibitor. We determined that geranic acid (GEA), which resembles the proposed enolate intermediate, acted as a non-competitive inhibitor of CrISY activity (K i ~ 4 μM) ( Supplementary Fig. 4), presumably by binding to the inactive CrISY/NADP + complex. Geranic acid could be co-crystallized with NADP + and the enzyme, allowing the structure of the ternary complex to be solved at 1.75 Å resolution (PDB code 5DF1) (Fig. 2b, Supplementary Table 1, Supplementary Fig. 3c,d).

Å resolution, Supplementary Figs. 1 and 2a and Supplementary
These three structures of CrISY (apo, NADP + , NADP + /geranic acid) were used to construct a model of catalysis. The absence of suitably placed cysteine or histidine residues substantiated our hypothesis that covalently assisted cyclization prior to reduction is unlikely. No lysine residue was correctly oriented for imine-assisted catalysis; although Lys146 is in the active site, its ε-amino group points away from the carboxyl moiety of the inhibitor.
The resemblance of negatively charged geranic acid to the proposed enolate intermediate supports the hypothesis that CrISY reduces 8-oxogeranial to an enolate. The backbone amide of Lys146 and/or the phenolic group of Tyr178 may bind to the C1 carbonyl of the substrate to stabilize the newly forming negative charge as reduction occurs (Fig. 2b). The backbone amide of Ile145 and the O-2' of the ribose of NADP + may also interact with the enolate to form an oxyanion hole (Fig. 2b) Fig. 13), suggesting no major role for this residue in catalysis. Activity of the Lys146 mutants argues against a tyrosine-lysine interaction that is proposed to modulate the pK a of tyrosine in short chain dehydrogenases. 18 Mutant CrISY Y178F showed a 400-fold decrease in k cat /K M and a 30-fold decrease in k cat (Supplementary Table 2) but still yielded cyclized product ( Supplementary   Fig. 7).
The oxyanion hole anchors geranic acid directly above the NADP + cofactor, an orientation in which the pro-S hydride of NADPH would be poised for addition to C3 (Fig. 2b). Such a hydride transfer would generate the S-stereocenter at this carbon, which is observed in the final product, further indicating a catalytically relevant orientation of the inhibitor. Overall, the ISY structures support reduction of 8-oxogeranial to the enolate followed by noncovalently assisted Michael cyclization.
The shape of the geranic acid binding pocket implies that the enolate intermediate is generated in a tightly packed linear orientation, which is not compatible with cyclization ( Fig. 2b, Supplementary Fig. 5). We assume that O1 of the enolate remains anchored in the oxyanion hole, which, after hydride transfer, is strengthened by the positive charge of the oxidized cofactor. Then, the enzyme must adopt a more open conformation to allow the intermediate to fold into the appropriate conformation to cyclize. Notably, the structure of NADP + bound CrISY reveals a mix of partially unresolved "open" and "closed" conformations involving the Gly150-Asp162 loop (Fig. 3a). In contrast, the inhibitor bound structure exists predominantly in the closed conformation, in which Phe152 locks the ligand in the linear conformation (Supplementary Figure 5). 19 We speculate that after formation of the enolate in the closed conformation, ISY rearranges to an open form to allow folding and cyclization.
To explore the catalytic relevance of the open and closed conformations, we biased the conformational equilibrium of the Gly150-Asp162 loop by mutating Gly150, the position at which the open and closed conformations bifurcate. Ramachandran plots show ( Supplementary Fig. 6) 20 that a residue other than glycine would favor the open conformation. The structure of CrISY G150A was solved with NADP + bound (PDB code 5DCY), proving that an open form of the loop is trapped by this mutation (Fig. 3b, Supplementary Fig. 14). CrISY G150A showed activity and product profiles comparable to wild type enzyme (Supplementary Table 2, Supplementary Fig. 7), though the K M did increase by five fold (Supplementary Table 2), suggesting that the closed loop conformation promotes substrate binding. Similarly, mutation of Phe152, which appears to lock the substrate in the linear conformation, causes a minor increase in K M (Supplementary Table 2, Supplementary Fig. 7). The activity of these mutants suggests that this conformational flexibility near the active site ( Fig. 3) is not essential for catalysis.
To validate that the open conformation could accommodate the cyclized product, we modeled iridodial into the G150A structure (Fig. 2c). The model indicates that a number of hydrophobic residues define the "open" binding pocket (Ile151, Leu203, Met213, Ile345). One side of the binding pocket is lined by a positive electrostatic potential (NADP +nicotinamide, Lys146, backbone amides) presumably stabilizing negative charge in the cyclizing enolate. The conformational flexibility at loop Gly150-Asp162 might allow both tight binding of the linear 8-oxogeranial and subsequent cyclization.
The factors that control substrate specificity in the P5βR family are not well understood. 21 While some P5βR enzymes from non-iridoid producing plants cyclize 8-oxogeranial, 22 the D. lanata P5βR that has been crystallized produces negligible iridodial or nepetalactol product ( Supplementary Fig. 7). Moreover, progesterone consumption by CrISY is barely detectable ( Supplementary Fig. 8). The inhibitor bound structure of CrISY suggests that the Ala343-Ser354 loop plays a role in substrate recognition ( Supplementary Fig. 2h). Ile347 of the DlP5βR sequence (Ala346 in CrISY) may clash with the linearly oriented 8-oxogeranial substrate, thus preventing binding ( Supplementary Fig. 9). Indeed, when the Ala343-Ser354 loop was swapped with the corresponding DlP5βR sequence, ISY activity of the chimera was diminished by 2000 fold (Supplementary Table 2) along with a substantial increase in progesterone turnover ( Supplementary Fig. 8). Additionally, the Phe149-Pro160 loop exhibits a change in structure compared to DlP5βR ( Supplementary Fig. 2e). Swapping this loop with the corresponding DlP5βR sequence improved progesterone reduction, while 8oxogeranial turnover was not greatly affected (Supplementary Figs. 7 and 8). These data suggest that the features controlling substrate selectivity are surprisingly subtle, making it challenging to predict function directly from sequence. Continued analysis of the physiological functions of the many P5βR homologues found in the plant kingdom 21 will provide further insights into the basis of substrate specificity in the P5βR family.
Structural analyses have greatly facilitated the engineering and directed evolution of canonical terpene synthases. 23,24 These structures of CrISY will set the stage for efforts to produce variants that can be used in synthetic biology, metabolic engineering and biocatalysis applications. Repurposing iridoid synthase based on mechanistic and structural information could unlock new monoterpene chemical space and also provide further insight into the balance between substrate reduction and cyclization.

Chemicals
NADPH and NADP + were purchased from Sigma-Aldrich. Carbenicillin was purchased from Formedium. Substrate 8-oxogeranial was synthesized as previously described. 6 All salts and solvents used in this study were of analytical grade unless otherwise specified.  The synthetic genes were amplified with primer combinations indicated in Supplementary  Table 4 and cloned into expression vector pOPINF to give pOPINF-CrISY, pOPINF-CrISY22dN, and pOPINF-DlP5bR. pOPINF-CrISY and pOPINF-CrISY22dN lack the codons for the C-terminal KAF tag present in pOPINF-CrISY22dNKAF but also encode residue Asn87 that differs from the uniprot entry. Protein CrISY 22ΔN expressed from plasmid pOPINF-CrISY22dN was used for crystallization of the NADP + /GEA complex. pOPINF-CrISY encodes all N-terminal residues of wild-type CrISY. Point mutations and swaps were introduced into the codon optimized gene by PCR. Mutation G150A was introduced, for instance, by amplifying a first fragment with primer pair CrISY22dN_synth_f/CrISY_F149_r and a second fragment carrying the mutation with primer pair CrISY_G150A_f/CrISY_synth_r. Both fragments were assembled in a second PCR reaction containing the fragments and the outer primers CrISY22dN_synth_f and CrISY_synth_r and cloned into the pOPINF vector. The resulting plasmid pOPINF-CrISY22dN-G150A was used to express protein for crystallization of the CrISY 22ΔN-G150A / NADP + complex. For the sake of simplicity, CrISY variants with the 22ΔN truncation and with or without C-terminal KAF tag are all referred to as 'CrISY' in the main text.
Protein expression and purification-Protein for the apo structure and the NADP + complex was expressed in strain Rosetta 2 (EMD Millipore) transformed with pOPINF-CrISY22dNKAF. All codon optimized genes were expressed in strain soluBL21 (DE3) (Genlantis). The pOPINF vector encodes an N-terminal His-tag that is cleavable with 3C protease. Cells were pre-cultured overnight at 37°C in LB medium containing 100 μg/mL carbenicillin. Aliquots of 1 mL each were used to inoculate two 2 L flasks containing 2YT medium and carbenicillin. When cultures reached an OD 600 of 0.5-0.8 after ca. 6 hours shaking at 37°C, protein production was induced by adding IPTG at a final concentration of 500 μM. Protein was expressed at 18°C for ca. 16 h. Cells were harvested by centrifugation, the supernatant was discarded and pellets were resuspended in 100 mL of 50 mM Tris-HCl buffer (pH 7.0) containing 300 mM NaCl, 1 mM DTT, one tablet of Complete EDTA free protease inhibitor (Roche), and 0.2 mg/mL lysozyme. The cells were disrupted by 7 min sonication on ice in cycles of 2 s sonication followed by a 3 s break. All subsequent steps were conducted at 4°C. The lysate was centrifuged for 20 min at 35,000 g and the supernatant containing the soluble protein was applied on a 5 mL HisTrap column connected to an Äkta Xpress purifier (GE healthcare). His-tagged protein was eluted with a linear gradient of 20 mM to 500 mM imidazole in 50 mM Tris/glycine buffer adjusted to pH 8.0 with hydrochloric acid and supplemented with 5% (v/v) glycerol, 0.5 M NaCl, and 1 mM DTT. Fractions containing the protein of interest were collected, incubated with 3C protease (120 μg) for 1 hour at room temperature, and concentrated to 2-3 mL in an Amicon 10 kDa MWCO centrifugal filter (Millipore). The concentrated solution was further purified by sizeexclusion chromatography on a Superdex 200 16/60 GF column (GE Healthcare). Fractions corresponding to the molecular weight of the CrISY dimer (83 kDa) were collected, combined, and washed with 20 mM Tris-HCl buffer (pH 8.0) containing 20 mM NaCl and 1 mM DTT (low salt buffer) in an Amicon 10 kDa MWCO centrifugal filter. After concentration to ca. 1 mL, protein was injected on a MonoQ 5/50 GL anion exchange column and eluted with a linear gradient of 20 mM to 1000 mM NaCl in low salt buffer. Fractions containing the protein of interest were combined and stored at −20°C after flash freezing in liquid nitrogen. For all mutants produced exclusively for kinetic characterization and DlP5βR, 3C cleavage and the final anion exchange chromatography step were omitted. Protein concentrations were determined spectrophotometrically at 280 nm using extinction coefficients calculated with the protparam tool (http://web.expasy.org/protparam/). A typical yield for CrISY produced with codon optimized genes on a 2 L scale was 4-5 mg after anion exchange chromatography. Proteins purified in this study were more than 95% pure as judged by SDS-PAGE ( Supplementary Fig. 12). Crystallographic methods-X-ray datasets were recorded on one of three beamlines at the Diamond Light Source (Oxfordshire, UK) (beamline 5DCU, I04; 5DCW, I04; 5DF1 I03; 5DCY, I04-1) at wavelengths of 0.9000-0.9795 Å (5DCU, 0.9000; 5DCW, 0.9000; 5DF1, 0.9795; 5DCY, 0.9173) using a Pilatus 6M detector (Dectris) with the crystals maintained at 100 K by a Cryojet cryocooler (Oxford Instruments). Diffraction data were integrated using XDS 26 and scaled and merged using AIMLESS; 27 data collection statistics are summarized in Supplementary Table 1. An initial model of the CrISY 22ΔN-KAF /NADP + complex was obtained by molecular replacement using PHASER 28 with the DlP5βR structure (PDB code 2V6G) 14 as the search model. Manual corrections were made in COOT 29 and the model was refined with REFMAC5. 30 As the crystals of the other two NADP + complexes belonged to the same space group (C222 1 ), their structures could be solved by refining against the CrISY 22ΔN-KAF /NADP + model. The structure of the apo complex, belonging to space group H32, was solved by molecular replacement with the CrISY 22ΔN-KAF /NADP + model. Ligand restraints were calculated on the Grade Web Server (http://grade.globalphasing.org/). In general, parameters for REFMAC5 refinement were optimized with PDB-REDO. 31 All models were subsequently refined with TLS group definitions obtained from the TLS-MD server. 32 Model geometries were validated with the MOLPROBITY 33 Fig. 11). 34 Although the C222 1 crystal form observed for all the holo CrISY structures contains two monomers in the asymmetric unit (ASU), these do not correspond to the biological dimer. Instead, the ASU comprises two halves of two discrete biological dimers. Each of these is generated through the application of a different two-fold crystallographic symmetry operator. Similarly, an equivalent dimer is generated by two-fold crystallographic symmetry from the single monomer in the ASU of the H32 crystal form observed for the apo CrISY structure. All possible structural superpositions were made within the resultant set of CrISY structures at both the monomer and dimer level, and the corresponding RMSD values are summarized in Supplementary Table 3. These showed that there was no significant variation in the relationship between the individual subunits within the biological dimers since the RMSD values calculated for the dimer superpositions were relatively low and not significantly larger than those calculated for the monomer superpositions. Similar values were observed for comparisons between the CrISY structures and those of DlP5βR. Moreover, the protein surface areas buried at the dimer interfaces were reasonably consistent, lying in the range 918 -979 Å 2 for CrISY and 888 -919 Å 2 for DlP5βR. All structural figures were prepared using PyMOL (PyMOL Molecular Graphics System, Version 1.3 Schrödinger, LLC.).

Protein crystallization-For
Enzyme assay by NADPH consumption-Kinetics of NADPH consumption were determined spectrophotometrically on a Perkin Elmer Lambda 35 instrument at a wavelength of 340 nm in cuvettes with 1 cm path length. Reactions contained 50 μM NADPH, 200 mM MOPS buffer pH 7.0, 100 mM sodium chloride and 2 nM to 4 μM enzyme, depending on the activity, in a total volume of 800 μL. 8-oxogeranial was added from a stock solution in inhibitor free THF. A co-solvent concentration of 1% was maintained in the assay to ensure solubility. Cuvettes were equilibrated to 25°C for 3 minutes before the reaction was initiated by addition of enzyme. Absorbance values were recorded at a rate of 1 Hz and initial velocities were linearly fit to at least 10 data points. with guard column for progesterone reactions. Helium was used as mobile phase at a constant flow rate of 1.0 mL/min and average velocity 36 cm/s. After 5 min at 60°C, the column temperature was increased to 150°C at a rate of 20 K/min, then to 280°C at 45 K/ min, and kept at 280°C for another 4 minutes. A solvent delay of 5 minutes was allowed before collecting MS spectra at a fragmentation energy of 70 eV.
Modeling-For modeling the iridodial 7,8-enolate (IEN) into the structure of CrISY 22ΔN-G150A /NADP + , the lowest energy conformer of the relevant stereoisomer of iridodial 7,8-enolate (IEN) was predicted in Chem3D Pro 13.0 (PerkinElmer). Initial coordinates for the IEN ligand were obtained by pair fitting atoms O1, C1, and C2 of GEA in the NADP + /GEA bound structure to the corresponding atoms in IEN. The resulting coordinates were then copied into the CrISY 22ΔN-G150A /NADP + model. The IEN bound structure was prepared for energy minimization by deleting water molecules and alternate side chain conformations from the model, and rotating the side chains of Lys146 and Ile145 to avoid steric clashes with the docked ligand using PyMOL. Hydrogens and charges were added to the ligands NADP + and IEN in UCSF chimera (IEN: -1). The resulting mol2 files were converted to params files readable by Rosetta with the molfile2params script. Then, the structure was energy minimized in Rosetta energy terms with a Rosetta relax script. 35

Circular Dichroism
Far ultraviolet (UV) CD spectra were recorded on a Chirascan Plus spectropolarimeter (Applied Photophysics) at 20°C in 10 mM potassium phosphate buffer pH 7.0. Protein concentrations were in the range 0.20-0.25 mg/mL. Spectra are presented as the CD absorption coefficient calculated on a mean residue ellipticity (MRE) basis.

Supplementary Material
Refer to Web version on PubMed Central for supplementary material. . 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. 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.