Structural Characterization of Early Michaelis Complexes in the Reaction Catalyzed by (+)-Limonene Synthase from Citrus sinensis Using Fluorinated Substrate Analogues

Abstract

The stereochemical course of monoterpene synthase reactions is thought to be determined early in the reaction sequence by selective binding of distinct conformations of the geranyl diphosphate (GPP) substrate. We explore here formation of early Michaelis complexes of the (+)-limonene synthase [(+)-LS] from Citrus sinensis using monofluorinated substrate analogues 2-fluoro-GPP (FGPP) and 2-fluoroneryl diphosphate (FNPP). Both are competitive inhibitors for (+)-LS with KI values of 2.4 ± 0.5 and 39.5 ± 5.2 μM, respectively. The KI values are similar to the KM for the respective nonfluorinated substrates, indicating that fluorine does not significantly perturb binding of the ligand to the enzyme. FGPP and FNPP are also substrates, but with dramatically reduced rates (kcat values of 0.00054 ± 0.00005 and 0.00024 ± 0.00002 s−1, respectively). These data are consistent with a stepwise mechanism for (+)-LS involving ionization of the allylic GPP substrate to generate a resonance-stabilized carbenium ion in the rate-limiting step. Crystals of apo-(+)-LS were soaked with FGPP and FNPP to obtain X-ray structures at 2.4 and 2.2 Å resolution, respectively. The fluorinated analogues are found anchored in the active site through extensive interactions involving the diphosphate, three metal ions, and three active-site Asp residues. Electron density for the carbon chains extends deep into a hydrophobic pocket, while the enzyme remains mostly in the open conformation observed for the apoprotein. While FNPP was found in multiple conformations, FGPP, importantly, was in a single, relatively well-defined, left-handed screw conformation, consistent with predictions for the mechanism of stereoselectivity in the monoterpene synthases.

Structural and Chemical Biology of Terpenoid Cyclases

“…Active site residues M458/I450 and N345/I336 of (−)-limonene synthase/(+)-limonene synthase appear to be the principal determinants of right-handed/left-handed helical conformations of the substrate leading to formation of the proper limonene stereoisomer. Reproduced from ref ( 195 ). Copyright 2017 American Chemical Society.…”

“…On the basis of analysis of enzyme complexes with fluorinated analogues, Oprian and colleagues suggest that active site residues M458/I450 and N345/I336 of (−)-limonene synthase/(+)-limonene synthase govern stereospecificity of the respective GPP cyclization cascades. 195 The Sδ atom of the M458 side chain would sterically clash with the terminal methyl groups of 2-fluorolinalyl diphosphate if it adopted an incorrect left-handed conformation in the active site of (−)-limonene synthase, and the Cγ2 group of I336 would similarly clash with the terminal methyl groups of 2-fluorogeranyl diphosphate if it adopted an incorrect right-handed conformation in the active site of (+)-limonene synthase ( Figure 39 ). This is an elegant demonstration showing how the active site contour of a terpenoid cyclase is a template that directs structure and stereochemistry in a cyclization cascade.…”

Structural Basis of Catalysis in the Bacterial Monoterpene Synthases Linalool Synthase and 1,8-Cineole Synthase

“… 45 , 58 Previous studies have indicated that some terpene cyclases/synthases can also accept neryl pyrophosphate (NPP) as substrate. 16 In the case of bCinS, incubation with NPP also leads to 1,8 cineole ( Figure 1 ). As observed with other terpene synthases, fluorination of the substrate blocks the key ionization step, blocking diphosphate release and formation of the geranyl/neryl cation.…”

“… 20 So far, bLinS and bCinS are the only characterized bacterial mTC(S) that accept GPP as substrate and thus lead to monoterpene formation. The bCinS-FNPP complex is specifically compared to the structures of plant limonene synthases 15 , 16 and bornyl diphosphate synthase 14 for which complexes with the substrate analogues are available. When comparing the corresponding C-terminal catalytic domains with the bCinS complexes, it is clear that the orientation of GPP/NPP analogue in bCinS is such that the beta phosphate occupies the location comparable to the alpha phosphate binding site in the plant enzymes and vice versa , and resembles the orientation observed in sesquiterpene synthase complex.…”

“…Data was found to be in accordance with the literature. 16 , 29 2-fluoronerol pyrophosphate : 1 H NMR (400 MHz, D 2 O) δ 5.14–5.06 (m, 1H, H7), 4.50 (dd, J = 23.6, 6.1 Hz, 2H, H1), 2.07 (br s, 4H, H5, H6), 1.62 (s, 6H, H4, H9/10), 1.54 (s, 3H, H9/10). 13 C NMR (126 MHz, D 2 O) δ 134.3 (s), 123.3 (s), 119.1 (d, J = 13.2 Hz), 60.4 (dd, J = 31.0, 4.3 Hz), 30.8 (d, J = 4.6 Hz), 25.7 (d, J = 2.7 Hz), 24.8 (s), 16.9 (s), 12.8 (d, J = 8.6 Hz) 31 P NMR (162 MHz, D 2 O) δ – 7.64 (d, J = 21.4 Hz), – 10.93 (d, J = 20.9 Hz) 19 F NMR (471 MHz, D 2 O) δ – 119.13 (t, J = 23.1 Hz).…”