Paeonia lactiflora, Pall.

Xu, Jingwei, Peng, Guanzu, Xu, Jinkun, Li, Yi, Tong, Li & Yang, Dong, 2021, Probing of the plasticity of the active site in pinene synthase elucidates its potential evolutionary mechanism, Phytochemistry (112573) 181, pp. 1-10 : 2-4

2.1. Homology modeling of P. lactiflora pinene synthase

The crystal structures of pinene synthases have not been reported. Therefore, we constructed a homolog model of P. lactiflora pinene synthase using the crystal structure of S-limonene synthase as the template. In order to investigate the catalytic mechanism, we docked three carbocation intermediates (terpinyl, pinyl and thujyl cations) into its active site ( Fig. 1B–D ). The docking results suggest that carbocations bind to a similar location in the active pocket. Nearby residues include R305, S311, W314, I335, I338, N339, D342, Y417, I443, A444, I448, F482, D486, S491 and H571. Some of these residues could make hydrophobic interactions with the carbon skeleton of carbocations. Certain polar or aromatic residues may stabilize the positive charge and influence the product profile of the enzyme.

2.2. Alanine scanning mutagenesis identifies mutations that cause an increase in sabinene production

We first identified residues within 5 Å from the docked carbocations and performed alanine-scanning mutagenesis on them. Most of these mutations do not show dramatic changes in their product profiles. However, two mutations show interesting alteration in the product profile. One of them, F482A, essentially converts the enzyme to sabinene synthase. About 90.8% of its products is sabinene, while 97.7% of the products of the WT enzyme is α- pinene ( Table 1 , Fig. 2A–B ). Another mutation, I335A, also causes an increase in sabinene production, though not as dramatic as F482A ( Table 1 ). Importantly, although both mutants show an altered product profile, their overall activities are only moderately decreased ( Table 1 ).

2.3. Phenylalanine at the 482 position is required for pinene production

We hypothesized that the aromatic ring of F482 might play a role in stabilizing the pinyl cation. To investigate this possibility, we mutated F482 to Val and Ile, two nonpolar residues with different side chain sizes. We also mutated it to Thr, Arg and Trp, representing polar, charged or non-Phe aromatic residues. The results suggest that any mutations that changes the phenylalanine at this position leads to an increase in sabinene production and almost abolishes the α- pinene production ( Table 1 ). Replacing phenylalanine with nonpolar residues (F482I, F482V) all effectively convert the enzyme to sabinene synthase ( Fig. 2C–D ). More than 90% of products made by these mutants are sabinene ( Table 1 ). Interestingly, F482T also predominantly produces sabinene, which seems to suggest that a polar residue here is not sufficient to stabilize the pinyl cation ( Table 1 , Fig. 2E ). Other mutations (F482Y, F482R and F482W) retain very little activities ( Table 1 ).

In order to verify that the mutant enzymes indeed produce sabinene, we analyzed the reaction product of F482L using the high resolution hybrid quadrupole-orbitrap GC-MS/MS system ( Fig. 3A–C ). By comparing with the sabinene standard, the peak eluted at 9.29 min was identified as sabinene. Its mass spectrum was measured and could be readily matched with that of sabinene standard ( Fig. 3B–C ).

It is interesting to note that replacement of F482 to other aromatic residues (Tyr or Trp) causes a drastic decrease in enzyme activities. To investigate this issue, we first docked the pinyl cation into the active pockets of WT and F482Y. It could be seen that in these two structures, the conformation of the docked pinyl cation is quite different ( Fig. 4A ). We then performed a 2 ns molecular dynamics simulation. The results suggest more conformational changes ( Fig. 4B–C ). Therefore, the extra hydroxyl group in Tyr could significantly change the conformation of the carbocation, which may explain the decreased activity and altered product profile.

2.4. S491A mutation enhances the overall activity of pinene synthase without changing its product profile

During our Ala scanning experiments, we identified a mutation S491A that enhances overall activities ( Fig. 5A, D , Table 1 ). The overall activity of S491A mutant is about 29% higher than that of the WT enzyme ( Table 1 ). Furthermore, S491A does not change the product selectivity, as more than 90% of their products remains α- pinene ( Fig. 5A , Table 1 ).

What is the mechanism of S491A to enhance the enzymatic activity? Since S491A still predominantly produces pinene, we docked the pinyl cation into the active pocket of the mutant enzyme and performed a 2 ns of molecular dynamics simulation. We also performed the same dynamic simulation on the WT enzyme in complex with the pinyl cation. Our data suggests that the active site residues in S491A have a much smaller RMSD than those in the WT enzyme, indicating the mutation S491A could cause the active pocket to be more rigid ( Fig. 6A , STable 2). Interestingly, simulation on the WT or S491A enzymes in complex with the terpinyl or thujyl cation does not show the same differences in RMSD values of active site residues ( Fig. 6B–C , STable 2). This probably indicates that the S491A mutation increases the rigidity of the active pocket only when it binds to the pinyl cation.

To better quantify the mobility of protein structures, we reanalyzed the simulation data using MDLovoFit ( Martínez, 2015). This way we could subdivide the protein into regions with high and low mobility ( Fig. 7A–C , STable 3). Interestingly, in S491A, fewer residues are classified as of high mobility than residues in WT enzymes (59% vs 63%). Furthermore, for residues classified with the highly mobile subset, those in S491A have lower average RMSD than those in WT proteins (0.253 nm vs. 0.278 nm). Therefore, there is a slight but significant increase in rigidity in S491A mutant protein.

2.5. Combination of S491A with F482 mutants has mixed results in the overall activity

Since all four sabinene synthase mutants show some decreases in the catalytic activity, we wondered whether we may restore their activities to that of the WT pinene synthase by combing with S491A. We produced double mutants F482A/S491A and F482I/S491A. Both seem to retain the same product selectivity of the original single mutants, though F482A/S491A shows an insignificant decrease in sabinene production (P =0.39; Fig. 5B–C , Table 1 ). In overall activities, F482I/S491A shows an insignificant increase in activity (P =0.27), while F482A/S491A has a significant decrease in activity (P <0.05; Fig. 5D , Table 1 ). Therefore, it seems that the effect of combining two mutants is more complicated than we expected.

We asked whether the combination of F482A and S491A mutations could affect the rigidity of the protein. After a 2 ns of molecular dynamics simulation and data analysis using MDLovoFit, we found the rigidity of F482A/S491A is similar to that of S491A (STable 3). Apparently, the decrease of activity by the combination of F482A with S491A cannot be explained by the overall RMSD change.

To further investigate this issue, we analyze the RMSF values based on the simulation data (SFig. 1A–C). Compared with S491A and WT, F482A/S491A shows an RMSF spike at a region between residues 219 and 223. In the structural model, this region forms a loop in the protein structural model (SFig. 2). However, this loop is quite far from the active site (about 40 Å). Therefore, its mobility could only affect the enzyme activity indirectly. For example, it could influence the closure of the active pocket.

We also superimposed the structural model of WT, S491A and F482A/S491A in complex with the docked pinyl cation (SFig. 3). In the structural model, the pinyl cation is docked between the residue 491 and 482 (SFig. 3). Interestingly, in WT and S491A, the position of the pinyl cation is similar, while in F482A/S491A, the pinyl cation is shifted towards the A482. This change in the carbocation position may impair the enzyme activity (SFig. 3).

Figures

Fig. 1. a, The proposed reaction mechanism for the synthesis of limonene, pinene and sabinene. b, The structural model of pinene synthase docked with the terpinyl (left), pinyl (middle) and thujyl (right) cations. Carbocations are colored in magenta (terpinyl), wheat (pinyl) and cyan (thujyl). Other atoms are colored according to their types (red: oxygen; yellow: carbon; blue: nitrogen; orange: phosphorus; green: magnesium). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 2. Converting pinene synthase to sabinene synthase by mutations on 482 position. Chromatograms in a, b, c, d and e show the GC-MS analysis of terpene products for WT, F482A, F482I, F482V and F482T, respectively. The x-axis is the retention time and the y-axis is the relative abundance of each species. The numbers in each peak correspond to α-pinene (1), sabinene (2) and limonene (3).

Fig. 3. Hybrid quadrupole-orbitrap GC-MS/MS. a shows the chromatogram of terpene products of F482L. The elution peak corresponding to sabinene is labeled. The mass spectrum of the product eluted at 9.29 min from a is shown in b. The mass spectrum of the standard sabinene is shown in c.

Fig. 4. Conformational differences in the pinyl cation in WT and F482Y as demonstrated by molecular dynamics simulation. The structures of WT and F482Y are superimposed. Results after 0, 1 and 2 ns of simulation are shown in a, d and c, respectively. Carbon in WT is colored in yellow and carbon in F482Y is colored in green. Oxygen is colored in red. Nitrogen is colored in blue. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 5. The effect of the S491A mutation. Chromatograms in a, b and c show the GC-MS analysis of terpene production for S491A, F482A/S491A, and F482I/S491A, respectively. The x-axis is the retention time and the y-axis is the relative abundance of each species. The numbers in each peak correspond to α-pinene (1), sabinene (2) and limonene (3). d shows the overall activity and the percentage of sabinene within total products produced by F482A, F482A/S491A, F482I and F482I/S491A. The asterisk indicates P <0.05.

Fig. 6. S491A mutation increases the rigidity of the active pocket when it binds the pinyl cation. RMSD values of the active site residues between WT and S491A in complex with the pinyl cation (a), terpinyl cation (b) and thujyl cation (c) are compared.

Fig. 7. The RMSD profiles of the region with high (RMSD H) and low (RMSD L) mobility from the simulation of WT (a), S491A (b) and F482/S491A (c) in complex with the pinyl cation.