Conversion of Substrate Analogs Suggests a Michael Cyclization in Iridoid Biosynthesis

Summary The core structure of the iridoid monoterpenes is formed by a unique cyclization reaction. The enzyme that catalyzes this reaction, iridoid synthase, is mechanistically distinct from other terpene cyclases. Here we describe the synthesis of two substrate analogs to probe the mechanism of iridoid synthase. Enzymatic assay of these substrate analogs along with clues from the product profile of the native substrate strongly suggest that iridoid synthase utilizes a Michael reaction to achieve cyclization. This improved mechanistic understanding will facilitate the exploitation of the potential of iridoid synthase to synthesize new cyclic compounds from nonnatural substrates.

, related to Figure 2. Steady-state kinetic analysis of the iridoid synthase reactions. A construct with a truncated N-terminus was used; this truncation has been used for all structural studies of the iridoid synthase homologue, progesterone beta reductase. Reaction rates were measured spectrophotometrically, monitoring NADPH consumption at 340 nm. Individual data points are averages of four replicates. Error bars represent standard errors (SE). The overlaid curves were obtained through non-linear regression (hyperbola), which yielded the rate equations inserted in each plot. (A) Saturation curve for 8-(difluoromethylene)geranial at a fixed NADPH concentration of 1 mM. K m = 485 ± 160 µM; k cat = 6.4 ± 0.8 s -1 .
Research. In case where no accurate mass spectra could be measured due to the instability of the compound, the standard GC-MS is provided for characterization.

C: Enzymatic assays
All enzyme assays were carried out using 20 mM MOPS pH 7.0 as buffer. The substrates were kept as 50 mM stocks in tetrahydrofuran (THF) at −20 °C. During the preparation of master mixes, care was taken not to exceed THF concentrations higher than 0.5% in the presence of enzyme, as concentrations above 1% THF were found to affect activity adversely. The batch of THF used contained a small amount of inhibitor of peroxide formation (butylated hydroxytoluene, BHT, added by the solvent manufacturer), which is visible as a small peak at approximately 10.35 min in the GC chromatograms of extracted enzymatic products.

Cloning and expression of truncated iridoid synthase
The USER-compatible vector pET28au was created by amplifying the GFP coding sequence from pEAQ-HT-GFP HindIII restriction sites. The coding sequence of iridoid synthase lacking 30 amino acids N-terminally was amplified from pDEST17-ISY [see reference 2 from main text] using primers GGCCTTAAUTGAAAACCTGTATTTTCAGGGCGTGGCACTAGTAGTAGGAGTCACC and affinity purification as described in reference 2 of the main text. Apart from six consecutive His residues, the N-terminal protein tag (MGSSHHHHHHSSGLVPRGSHMASMTGGQQMGRGSAEALIENLYFQG) also provided a TEV cleavage site (NLYFQG), but all attempts to remove the tag using TEV protease were unsuccessful.
Amino acid sequence of iridoid synthase. Residues crossed out were deleted in the truncated construct.:
The reaction was monitored continuously by TLC using a short-wave ultraviolet lamp for detection, and went to completion after 1 h. The products were extracted with dichloromethane ( ppm. 13 C NMR ( It should be mentioned that the molecule 6 decomposes in aqueous solution within approximately 3 hours. However, time courses showed that the enzymatic reaction is complete within several minutes. Since the enzymatic reaction is so much faster than the decomposition, mechanistic studies were not affected.

Spectrophotometry-based assays (kinetic and substrate specificity studies)
For kinetic studies, the absorbance at 340 nm of 200 µL assays was measured using a 96-well plate reader. See the legend to Supplementary Fig.5 for further experimental details. Data were collected for 3-15 min, with individual measurements being taken for a single sample every 10 s when using full plate. The measured absorbances were plotted manually on Excel and the initial delta absorption/delta time values were calculated for the linear part of each reaction. NADPH consumption rates were calculated from these delta absorption/delta time values considering background NADPH decay and an extinctioncoefficient-like value (dependent on assay volume) calculated separately. The Michaelis-Menten curve was fitted using Origin software.