Biocatalytic routes to stereo-divergent iridoids

Abstract

Thousands of natural products are derived from the fused cyclopentane-pyran molecular scaffold nepetalactol. These natural products are used in an enormous range of applications that span the agricultural and medical industries. For example, nepetalactone, the oxidized derivative of nepetalactol, is known for its cat attractant properties as well as potential as an insect repellent. Most of these naturally occurring nepetalactol-derived compounds arise from only two out of the eight possible stereoisomers, 7S-cis-trans and 7R-cis-cis nepetalactols. Here we use a combination of naturally occurring and engineered enzymes to produce seven of the eight possible nepetalactol or nepetalactone stereoisomers. These enzymes open the possibilities for biocatalytic production of a broader range of iridoids, providing a versatile system for the diversification of this important natural product scaffold.

Fig. 1. Overview of the nepetalactol scaffold and its stereo-control in the Nepeta species.

A The early iridoid pathway takes two main directions based on the preferred stereochemistry of iridoid synthase (ISY). 7S-ISY leads to biosynthesis products such as (7 S) nepetalactones found in most Nepeta species, as well as well-known indole alkaloid precursor strictosidine in C. roseus and other species (red). 7R-ISY exists in other members of the Asterids clade, particularly the Lamiales order, where iridoid glucosides with this C7 stereochemistry is common (light blue). B Nepetalactol 3 stereochemistry can adopt 8 stereo-chemical configurations based on the relative positions of the C7 methyl group and the 7a-4a bridge. Most iridoids known in plants are based on the 7S-cis-trans 3a and 7R-cis-cis 3b’ configurations (7 S, 4aS, 7aR and 7 R, 4aS, 7aR, respectively). C Three Nepeta species studied here, N. sibirica, N. mussinii, and N. cataria. D As for nepetalactol 3, nepetalactone 4 also can exist in 8 stereo-chemical configurations. NEPS typically control the stereochemistry of the bridgehead carbons; in one instance a Major Latex Like Protein catalyzes the formation of 7S-cis-trans 3a (7 S, 4aS, 7aR). NEPS also must be capable of cyclization and oxidation of both the 7 S and 7 R products of ISY in a selective manner to generate the full complement of stereoisomers.

Fig. 2. Decoupling of cyclization and dehydrogenation of 7S-cis-trans and 7S-cis-cis activities.

A N. sibirica NEPS2 (NsNEPS2) oxidation activity can be readily decoupled from the cyclization activity with catalytic Y167F amino acid substitution to produce 7S-cis-trans nepetalactol 3a. Peaks marked with * represent iridodial 5 side products. B NcNEPS3A and NmNEPS3 are highly similar enzymes but only NcNEPS3A can oxidize 7S-cis-cis nepetalactol 3b. NmNEPS3-Q206V mutant has restored oxidation activity and NcNEPS3A-V206Q has diminished oxidation activity showing that this residue is directly involved in the binding of 7S-cis-cis nepetalactol 3b to enable oxidation. C Molecular docking of 7S-cis-cis nepetalactol 3b suggests that Q206 in NmNEPS3 could be negatively interacting with the cyclo-pentane ring in the nepetalactol and preventing binding. Highlighted parts of chromatograms represent the molecular structure highlighted with the same color. Results were repeated three times independently with similar results.

Fig. 3. Decoupling of cyclization and dehydrogenation of 7S-trans-cis activity.

A Existing enzymes have only partial roles in cyclization (NmNEPS4) and oxidation (NmNEPS1) of 7S-trans-cis nepetalactol 3c. B NmNEPS1 was engineered to perform both cyclization and oxidation to form 7S-trans-cis nepetalactone 4c. The best variant found (NmNEPS1-154SVTA) is able to produce significantly more 4c than either NmNEPS1 or NmNEPS4 alone. In addition, L199 residue in NmNEPS4 appears to be involved in destabilizing the 7S-trans-cis nepetalactol 3c. C Molecular docking of 3c showing the 154SATA loop and L198 suggesting these residues might clash with the C4 methyl group in 3c, preventing proper positioning for oxidation. Highlighted parts of chromatograms represent the molecular structure highlighted with the same color. Results were repeated three times independently with similar results.

Fig. 4. Nepeta sibirica ISY, NEPS and MLPL screening.

A Chiral GC-MS traces show that both NsISY and NsP5ßR have 7S-ISY activity, as evidenced by the alignment of the products (7S-cis-trans nepetalactol 3a and iridodials 5) with those of the 7S-specific CrISY and not the 7R-specific LaISY. The red dashed line represents the 7S-cis-trans nepetalactol 3a peak and the peaks marked with * represent iridodial 5 side products. B Phylogenetic tree of all NEPS from N. sibirica found and their relationship with N. mussinii, and N. cataria. C Relative expression of NEPS in N. sibirica shows that young leaves tissue contains the highest expression profile and NsNEPS1A is the most highly expressed of the NEPS. D Enzymatic activity of N. sibirica enzymes. NsNEPS as well as NsMLPL identified in combination with identified NsISY shows the various nepetalactone profiles obtained. Most notably, NsNEPS1B produces a significant amount of 7S-trans-trans nepetalactone 4d. E Scheme of activities found by combining NsISY and various NsNEPS and NsMLPL enzymes. Highlighted parts of chromatograms in part D represent the molecular structures highlighted with the same color in part E. NEPS names in parts B, C, and D, are color coded. Results were repeated twice independently with similar results.

Fig. 5. Nepeta sibirica produces both 7 R and 7 S iridoids.

A The 7 S specific ISY comes from C. roseus while the Lamium album iridoid synthase (LaISY) yields the 7 R stereoisomer 2'. B GC-MS traces using a chiral column capable of separating 7 S (yellow highlight) and 7 R (light blue highlight) enantiomers. Peak indicated with * in N. sibirica leaf trace corresponds to germacrene. Surprisingly, N. sibirica leaves produce largely 7 R nepetalactones with 7 S nepetalactones as a minor product. When NsNEPS1A and NsNEPS1B are tested with LaISY the resulting products match those found in the plant. C DBU based epimerization scheme to confirm the C7 stereochemistry of N. sibirica trans-trans nepetalactone. Using previously characterized 7S-cis-cis nepetalactone 4b from N. mussinii and converting it to 7S-trans-trans 4d it is possible to compare both molecules using chiral column GC-MS (part B). D Circular dichroism spectra was used to distinguish the nepetalactone enantiomers. Results were repeated twice independently with similar results.

Fig. 6. Combinatorial biosynthesis for the production of 7 R nepetalactones.

A Various NEPS and MLPL enzymes were combined with LaISY in order to access 7 R nepetalactones. No NEPS combination produced 7R-trans-cis nepetalactone 4c’. B Similarly to 7S-cis-trans nepetalactone 4a (Fig. 2A), NsNEPS2 and NsNEPS-Y167F are capable of effectively producing 7R-cis-trans nepetalactone 4a’ and 7R-cis-trans nepetalactol 3a’, respectively. Peaks marked with * represent iridodial 5 side products. C A combination of NsMLPL1 and NsNEPS1B generated 7R-cis-cis nepetalactone 4b’ as a minor product. D Our best 7S-trans-cis nepetalactone producer, NmNEPS1-154SVTA is capable of high production of 7R-trans-trans nepetalactone 4d’. Highlighted parts of chromatograms represent the molecular structure highlighted with the same color. E Depiction of how NEPS have different ways to control the stereochemistry of cyclization. NsNEPS2 is able to catalyze cyclization of enantiomers yielding cis-trans stereochemistry for both 7 S and 7 R ISY. NmNEPS-154SVTA and NmNEPS3-206V appear to have specificity for the absolute configuration of the bridgehead carbons, and the orientation of the C7 methyl group does not change it. Results were repeated three times independently with similar results.