Abstract
Many functionally promiscuous plant 2,3-oxidosqualene cyclases (OSCs) have been found, but complete functional reshaping is rarely reported. In this study, we have identified two new plant OSCs: a unique protostadienol synthase (AoPDS) and a common cycloartenol synthase (AoCAS) from Alisma orientale (Sam.) Juzep. Multiscale simulations and mutagenesis experiments revealed that threonine-727 is an essential residue responsible for protosta-13 (17),24-dienol biosynthesis in AoPDS and that the F726T mutant completely reshapes the native function of AoCAS into a PDS function to yield almost exclusively protosta-13 (17),24-dienol. Unexpectedly, various native functions were uniformly reshaped into a PDS function by introducing the phenylalanine → threonine substitution at this conserved position in other plant and non-plant chair-boat-chair–type OSCs. Further computational modeling elaborated the trade-off mechanisms of the phenylalanine → threonine substitution that leads to the PDS activity. This study demonstrates a general strategy for functional reshaping by using a plastic residue based on the decipherment of the catalytic mechanism.
The combination of computational and experimental techniques shows a uniform functional trade-off of 2,3-oxidosqualene cyclases.
INTRODUCTION
Triterpenoids are one of the largest groups of natural products with numerous pharmaceutical applications (1–3). 2,3-Oxidosqualene cyclases (OSCs; also known as triterpene synthases) are a unique enzyme family with more than 250 known isoforms that can catalyze the common, acyclic substrate to form multiple triterpenes products. They serve as key enzymes that are responsible for the cyclic skeleton diversity of triterpenoids. According to an earlier report, approximately 200 distinct carbon skeletons had been found (4), and to date, the number has increased to more than 300 in view of the most recent terpenoid database (5). Among them, several types of triterpenoids such as cycloartenol and lanosterol are produced along with a total of more than a thousand other structures (see Fig. 1). The protostadienol-type tetracyclic triterpenes are rarely found in nature and their distribution is primarily limited to the genus Alisma of the Alismataceae family (6), and the enzyme responsible for its biosynthesis has been rarely reported. The only previously reported enzyme is a promiscuous OSC from fungus Aspergillus fumigatus (AfPDS) that yields protosta-17 (20),24-dienol (7, 8) as the major product, going through a chair-boat-chair (CBC) conformation of the first three fused rings of the tetracyclic protosteryl cation (Fig. 1) (9). It is well known that the CBC conformation triterpenes including cycloartenol, lanosterol, cucurbitadienol, parkeol, and others are mostly produced by undergoing a cascade of rearrangements from the protosteryl cation.
Fig. 1. The typical triterpenoids derived from CBC conformation protosteryl cation.
The carbon-skeleton differences are highlighted by magenta, and the number of natural products with each carbon skeleton is given in parentheses. The only difference between the two rarely reported protostadienol compounds is the C═C double bond at C17.
In this work, a unique plant source protostadienol-type tetracyclic triterpene cyclase (AoPDS) with a common cycloartenol synthase (AoCAS) was first characterized from Alisma orientale (Sam.) Juzep in a yeast heterologous expression system. Computational simulations of the enzymatic mechanism and experimental mutagenesis found a plastic substitution F726T of AoCAS that could reshape its native function to yield the protostadienol product. Further experiments found that the uniform trade-off from native activity to protostadienol synthase (PDS) activity occurs widely in several other CBC-type OSCs (catalyzing the formation of CBC conformation products) by a single F → T substitution at the same position. Last, the mechanism insights of functional trade-off were revealed by combined quantum and molecular mechanics (QM/MM) calculation.
RESULTS AND DISCUSSION
Characterization of AoCAS and AoPDS from A. orientale (Sam.) Juzep
A. orientale (Alismataceae) is a traditional and widely used Chinese medicinal herb native to several Asian countries, including China, Korea, and Japan. Protostane tetracyclic triterpenoids are thought to be chemotaxonomic markers of the genus (6). Two candidate unigenes AoOSC1 and AoOSC2 were screened from the publicly available transcriptome sequence data of A. orientale (National Center for Biotechnology Information, accession no. PRJNA417185). These two unigenes were cloned into the express vector pESC-Ura and transformed into a high-yield squalene yeast engineering strain SE-MET (see full experimental procedures in the Supplementary Materials). AoOSC2 (AoCAS) exclusively produces cycloartenol, whereas AoOSC1 (AoPDS) only produces protosta-13 (17),24-dienol (Fig. 2 and figs. S1 to S3), the precursor of alisol compounds [such as alisol B 23-acetate (10, 11) with hypolipidemic and anticancer activity]. AoOSC1 was the first characterized plant source PDS with a typical double bond at 13 (17), while AfPDS from fungal protosta-17 (20),24-dienol was the only known PDS.
Fig. 2. Characterization of AoCAS and AoPDS.
AoCAS and AoPDS were characterized as CAS and protosta-13 (17),24-dienol synthase, respectively. The mass spectra of the standard cycloartenol are shown in fig. S16. m/z, mass/charge ratio.
Identification of T727/F726 responsible for AoPDS and AoCAS product specificities
To decipher the different catalytic functions of AoPDS and AoCAS that share high sequence identity (>79%), we first performed QM/MM multiscale simulations to probe the catalytic mechanism from the common protosteryl cation. The plausible reaction pathways are proposed in Fig. 3A, starting from the protosteryl cation, followed by consecutive rearrangements including hydride shifts and methyl transfers, and finally deprotonation that leads to cycloartenol in AoCAS, while early deprotonation from either A or B state produces protosta-13 (17),24-dienol in AoPDS. The free energy profiles from protosteryl cation to the D state in AoCAS and protosta-13 (17),24-dienol in AoPDS were determined by QM/MM molecular dynamics (MD) simulations. The reaction energetic profiles are summarized in Fig. 3B, and corresponding structures for reaction intermediates are shown in Fig. 4.
Fig. 3. QM/MM multiscale simulations of catalytic processes.
(A) The plausible reaction pathways of AoPDS and AoCAS from the protosteryl cation. (B) Free energy profiles of AoPDS (red), AoCAS (blue), and AoCAS-F726T (dashed green).
Fig. 4. Representative structures of reaction intermediates in AoCAS/AoPDS.
The transition state structures are shown in figs. S8 and S9.
For the catalytic reaction in AoCAS, the 1,2-hydride shift in the protosteryl cation leads to the A state with a low barrier of 3.7 kcal/mol (Fig. 3B). The A state is unstable and is further converted to the B state through a different hydride shift with very low barrier (~1 kcal/mol). The next two steps involve 1,2-methyl transfers from the B state to D state (fig. S4) that also cross very low barriers (<3 kcal/mol). During the reactions, the aromatic side chain of F726 faces the D ring of the tetracyclic intermediate to stabilize the carbocation through cation-π interactions (Fig. 4). Accordingly, AoCAS is a high-fidelity OSC responsible for the exclusive production of cycloartenol, as validated by our further experiments (see Fig. 2).
As for AoPDS, the 1,2-hydride shift energy barriers (7.7 and 3.8 kcal/mol) are much higher than those in AoCAS, and as a result, the premature deprotonation from A or B state occurs with T727 acting as a general base in AoPDS, as shown in Fig. 4. The T727 in AoPDS is located at the same position as the aromatic F726 in AoCAS. We found a water hydrogen bond chain near the T727 and stably maintained during the MD simulations (Fig. 4). This may be due to the replacement of the hydrophobic residue F to the polar residue T. Although the hydroxyl group of T is not often regarded as a strong general base, the pKa (where Ka is the acid dissociation constant) of a protonated alcohol is typically around −1 to −4 (10), and the formation of water chain will further increase the alkalinity of T727 and facilitate the proton transfer from intermediates (C13H of A state and C17H of B state; see Fig. 3A) to T727; thus, it is reasonable to regard T as the general base [T as a general base has also been discussed in previous studies (11, 12)]. The deprotonation from C17H of B state with 6.1 kcal/mol barrier (Fig. 3B) is preferable to the C13H of A state because of its barrier (13.8 kcal/mol) (fig. S5). Therefore, the most plausible transformation from protosteryl cation to protosta-13 (17),24-dienol in AoPDS is a premature deprotonation at intermediate B by T727. Accordingly, protosta-13 (17),24-dienol is the only product of AoPDS detected in our experiments. Although several different amino acid residues do exist in the active pockets of these two enzymes, the above computational modeling verified that the F (or T) in the corresponding position of 726 (or 727) is probably the main difference in the enzymatic reactions catalyzed by AoCAS and AoPDS.
The strong trade-off effect of AoCAS-F726T from native activity to produce protosta-13 (17),24-dienol
To confirm the key catalytic roles of T727/F726 in AoPDS/AoCAS, site-directed mutagenesis experiments were carried out and the product profiles are summarized (Fig. 5 and figs. S6 and S7). Notably, F726T of AoCAS reshapes its native function into the PDS function almost completely [99.7% protosta-13 (17),24-dienol]. Other F726 substitutions to residues with hydroxy group side chain such as T (including F726Y and F726S) will become inactive and not produce protosta-13 (17),24-dienol, while most of the AoPDS mutants, especially substitutions of the T727 position, exhibited very low activity or even inactive, thus proving the essential deprotonation role of T727 in AoPDS. The activity could be maintained to some extent in the AoPDS-T727S mutant, which is likely because of S being somewhat similar to T with comparable side chain lengths and hydroxy groups, thus further confirming the aforementioned catalytic mechanism (Figs. 3 and 4) that T727 is the key residue for production of protosta-13 (17),24-dienol in AoPDS. Regarding the other different residues in the AoPDS/AoCAS active site besides T727/F726, substitutions of V411/Y410 and V482/I481 show more or less activity loss (Fig. 5), indicating that these two positions were not related to the deprotonation and protosta-13 (17),24-dienol production. The product spectrum is sharply altered only by the F726T substitution in AoCAS. Further QM/MM MD simulations on the AoCAS-F726T mutant showed that the reaction energy profile is very similar to that of the wild-type AoPDS (Fig. 3). Therefore, T727 is the key residue, as the general base for the premature deprotonation of the active carbocation, to yield protosta-13 (17),24-dienol exclusively in AoPDS. Notably in AoCAS, F726 is a plastic residue to harness the catalytic function of AoCAS from the native product spectrum of cycloartenol to protosta-13 (17),24-dienol by a F726T single substitution. As far as we know, such a strong trade-off effect with high catalytic efficiency and specificity for a new function by a single-residue substitution has not been previously reported for the large OSCs enzyme family.
Fig. 5. The product variations of AoPDS/AoCAS mutations.
The relative proportion of mutations was calculated by comparing with corresponding wide-type AoPDS/AoCAS that were set as 100%. The error bars were from three dependent mutation experiments.
The uniform functional trade-off of the typical CBC-type OSCs
Inspired by the functional reshaping in AoCAS-F726T and considering that F726 is highly conserved in most CBC-type OSCs (Fig. 6A and figs. S10 and S11), the functional plasticity of the F in other OSCs was further investigated, including ItOSC3 from Iris tectorum Maxim (5), SgCBQ from Siraitia grosvenorii (13), AjPS from Apostichopus japonicus (14, 15), and ERG7 from Saccharomyces cerevisiae (16), as all these OSCs have F at the same site as F726 in AoCAS.
Fig. 6. The functional trade-off of typical CBC-type OSCs.
(A) Sequence alignment analysis of reviewed OSCs (70 in total) and all available OSCs (256 in total including unreviewed sequences). (B) F → T substitution at same position as AoCAS in other CBC-type OSCs; the products of wild-type OSCs and their mutants are shown in dashed boxes and the circle, respectively [ItOSC3, SgCBQ, and AjPS was done in this work and ERG7 was previously reported (16)]. (C) Comparisons of wild-type and F → T mutant models of OSCs.
For the strong trade-off effect of the F → T substitution, the native catalytic function of all the above four OSCs were consistently reshaped into PDS function to produce protostadienol. As summarized in Fig. 6B and fig. S12, the ItOSC3, AjPS, and ERG7 mutants were converted into PDS function almost completely (96.7, 90.7, and 99.8% protostadienol, respectively), and the SgCBQ mutant shows a nearly half production proportion (49.5%). Sequence alignment of these selected CBC-type OSCs with AoPDS/AoCAS shows that the conversion to PDS function through the mutation is not limited by a certain level of sequence identity (fig. S17), because the AjPS and ERG7 with relatively low sequence identity (~37 and 34%, respectively) and active site similarity (both with more than six residue differences) to AoPDS still produce high percentage of protostadienol by the F → T substitutions. To decipher the intrinsic mechanism behind the high plasticity of the conserved F in these OSCs, QM/MM MD simulations were used to observe the dynamic interaction modes between F/T and the protosteryl cation (Fig. 6C). In wild-type enzymes, the protosteryl cation is stabilized by the aromatic F through cation-π interactions, which widely exists as a selectivity control factor in terpene synthases (17–20). As for the subsequent rearrangements from the protosteryl cation, CH-π interaction between C17 and F726 stabilizes the transition state to promote the 1,2-hydride transfer (C17 to C20); otherwise, it would take the route with the higher barrier as seen in AoPDS and previously studied hLAS F696 mutants (16, 18). While for the F → T mutants, the T supplements its hydroxy group to attract a proton from C17 or C13, causing the reactive carbocation to be quenched from A or B states. That is, the strong trade-off effect of F → T mutants is due to the supplemental deprotonation role of T, which replaces the original stabilizing contribution of F. Considering that the plasticity is commonly exhibited in the above four OSCs (all have the conserved F) and the high conservatism among CBC-type OSCs, we deduce that the specific F → T mutants are widely feasible (at least for those OSCs that yield tetracyclic products) for harnessing the new catalytic function to effectively biosynthesize the rare protostadienol.
The diverse trade-off effects of the same plastic position in chair-chair-chair–type OSC
Considering that the plastic F in CBC-type OSCs is also highly conserved in CCC-type (namely, chair-chair-chair conformation of the first three fused rings) OSCs, the potential plasticity of F in the CCC-type OSCs was also investigated. We found that the stabilization/quenching trade-off effect by F → T substitution is not well reproduced in CCC-type OSCs, which was proved by several typical OSCs with different products (figs. S13 to S15). Similar to GgBAS (Fig. 7A), the activity was largely decreased and only trace amount of dammarenediol II was produced in the F728T mutant. Another reported substitution producing dammarenediol-II (oat β-amyrin synthase S728F) (21) was also tested, and the GgBAS-N731F mutant did not obviously change the product spectrum. Instead, another notable trade-off between wild-type β-amyrin and abortive pentacyclic quenching activity was detected in the GgBAS-F728S mutant (other hydroxy group side chain residues were further attempted by F728S and F728Y mutants) and produces germanicol as the major product. QM/MM MD simulation shows that the S728 closes to the E ring of the oleanane-type intermediate (especially C18 and C19) for deprotonation rather than the D ring (Fig. 7B). Because mutants of the same position bring nonuniform enzymatic activity in CCC-type OSCs and the functional trade-off is not as strong as observed in CBC-type OSCs, the diverse trade-off effects found in CCC-type OSC mutants might also be a good initial point for functional reshaping but requires further extensive studies. In addition, similar protostadienol products are yielded both in AoPDS (this work) and in previously reported AfPDS; but as obviously shown in Fig. 7C, the AoPDS has high similarity with plant CAS and is unique from the AfPDS, which is closer to fungal lanosterol synthase (LAS). This reminds us that the mutant of AoCAS/ItOSC3 and ERG7 (also belong to CAS or LAS family, respectively) shows much higher protostadienol product rate (>96%) than that in others (such as SgCBQ; <50%), as shown in Fig. 6B. Considering that F is highly conserved at the same position while the appearance of T is unique (only found in AoPDS), the plastic F → T substitution likely causes a universal activity trade-off in many other CBC-type OSCs especially for CAS and LAS in plant.
Fig. 7. The functional trade-off of typical CCC-type OSC GgBAS.
(A) Product profiles of wild-type GgBAS and mutants. F728S mutant shows promiscous product spectrum with germanicol as the main product. (B) The model of GgBAS F728S mutant with 6-6-6-6-6 oleanane-type intermediate. (C) Phylogenetic tree of characterized plant OSCs.
In this work, inspired by deciphering the enzymatic catalytic mechanism for the unique A. orientale oxidosqualene cyclases (AoPDS and AoCAS), we found that the specific F → T substitution is a controllable factor to extensively produce protostadienol in CBC-type OSCs. It is remarkable that the uniform functional trade-off could be facilely realized by a single substitution, which will be instructive for other enzyme engineering, by combing multiscale simulations to reveal the plastic position with mutagenesis experiments to further validate its feasibility.
MATERIALS AND METHODS
Plant material and microbial strains
Ten- to 12-month-old A. orientale (Sam.) Juzep. (Alismataceae) plant was collected from JianOu, FuJian province, China. The plant was confirmed to be A. orientale belonging to the Alismataceae family by A. Ji (Guangzhou University of Chinese Medicine) and was maintained in the constant temperature incubator of Guangzhou University of Chinese Medicine. Escherichia coli DH5α was purchased from TransGen Biotech Co. Ltd. (Beijing, China). The S. cerevisiae strain SE-MET was constructed in our previous research.
Cloning of OSCs and construction of yeast expression plasmids
The full-length coding sequences were present for AoOSC1 (AoPDS) and AoOSC2 (AoCAS) in the transcriptome databases. These were directly amplified from the cDNA of A. orientale rhizomes using the Phanta Max Super-Fidelity DNA Polymerase (Vazyme Biotech Inc.) with gene-specific primers (table S4). Conditions for polymerase chain reaction amplification were as follows: 95°C for 3 min, (95°C for 15 s, 57°C for 15 s, 72°C for 2 min) × 32 cycles, and 72°C for 5 min. The resulting amplicons were constructed into the pESC-Ura vector (Agilent Technologies Inc.) between the Bam HI and Sma I restriction sites using the ClonExpressII One Step Cloning Kit (Vazyme Biotech, Nanjing, China) and validated by sequencing.
Phylogenetic and amino acid sequence analysis
The deduced amino acid sequences of AoPDS, AoCAS, and those of other OSCs (table S1) obtained from GenBank were subjected to phylogenetic analysis. All OSC proteins were aligned by ClustalW (22). The analysis was performed using the neighbor-joining method with MEGA 6.0 (23). Bootstrapping with 1000 replicates was used to estimate the strength of the nodes in the tree (24). Sequence similarities and secondary structure information of aligned sequences were rendered using ESPript 3.06 (25) based on the secondary structure of the human oxidosqualene cyclase (lanosterol cyclase) 1W6K, the only OSC for which experimentally determined structure information is available (26).
Metabolite extraction and gas chromatography–mass spectrometry analysis
Product analysis was collected by centrifugation of yeast cell biomass after fermentation. Transgenic yeast cells harboring the AoPDS and AoCAS genes were saponified in 2 ml of saponification reagent [20% (w/v) KOH in 50% (v/v) ethanol] at 100°C for 5 min and extracted three times with an equal volume of hexane, combining organic phases and evaporating under reduced pressure to obtain the crude extract. Crude extracts were derivatized by treatment with 50 μl of N-methyl-N-trimethylsilyl-trifluoroacetamide at 90°C for 10 min and then analyzed using a gas chromatography–mass spectrometry (GC-MS) equipment. Three biological replicates were performed for each sample.
Purification of protosta-13 (17),20-dienol (the product of AoPDS)
Seven liters of S. cerevisiae SE-MET cells expressing AoPDS were cultured and collected by centrifuging the culture at 5000 rpm. Saponification was carried out in 1 liter of saponification reagent (3.57 M KOH with 10.87 M ethanol) and refluxed at 75°C for 3 hours. The products were extracted four times with an equal volume of hexane. The hexane extract was loaded onto a silica gel column (30 cm by 2.5 cm, 200- to 300-mesh particle size; Haiyang, Qingdao, China) and eluted with hexane:ethyl acetate = 15: 1 (v/v). Fifty-milliliter fractions were collected and analyzed by thin-layer chromatography and GC-MS. Full experimental details appear in the Supplementary Materials.
Computational modeling and QM/MM MD simulations
AlphaFold2 (27) was used for OSCs modeling, the protosteryl cation intermediate after cyclizations was used as the starting point for CBC OSCs system setup and subsequent computational studies, and the dammarenyl cation intermediate was used for CCC GgBAS. The computational models were set up for further MD simulations to relax the structures of the proteins and the intermediates. Snapshots of each system from the stable trajectories were chosen to build the initial structures for the subsequent QM/MM simulations. The periodic boundary condition was also considered in the following QM/MM MD simulations. For AoPDS/AoCAS, the protosteryl cation and T727/F726 were included in the QM region, respectively (the charge of the QM region is 1). The same QM regions were used for ItOSC3, SgCBQ, AjPS, and Erg7. For GgBAS, the dammarenyl cation and F728 were included in the QM region. In all mutant models, the QM regions were kept the same as in the wild-type enzymes. All of these QM atoms were described with the M06-2X (28, 29)/6-31G(d) basis set, which is widely used in studying cyclization reactions (19, 30), and the model contains more than 730 basis functions in total. All of these QM/MM calculations were performed with the interfaced QChem (31)–AMBER12 programs (32). Full computational details appear in the Supplementary Materials.
Acknowledgments
We thank the Guangzhou and Shenzhen Supercomputer Center for providing computational source. We also thank Y. Zhang and S. g. Wang at NYU, as well as Y. Zhou at NJU, for help using the QChem-AMBER program.
Funding: This research was supported by the Key-Area Research and Development Program of Guangdong Province (2022B1111080005 to R.W.), the National Natural Science Foundation of China (22103098 to F.Z., 81874333 to L.D., 81930114 to Z.L., and 21773313 to R.W.), and the Postdoctoral Research Foundation of China (2020TQ0390 and 2020M683144 to F.Z.).
Author contributions: R.W. and L.D. supervised the whole research. R.W. designed the computational subproject. L.D. and Z.L. designed the experimental subprojects. R.Z. and Y.-e.H. isolated and identified the products. J.Y. and R.H. analyzed the sequences and data. A.-j.J. identified the plants and did the bioinformatic analysis. F.Z., R.W., and L.D. wrote the manuscript. B.A.H. and Z.L. made important contributions on revising the manuscript. F.Z. contributed many concepts and most computational simulations. Y.W. performed the experiments and data analysis. All authors contributed to the interpretation of the results. All authors reviewed and approved the final manuscript.
Competing interests: The authors declare that they have no competing interests.
Data and materials availability: All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials.
Supplementary Materials
This PDF file includes:
Supplementary Text
Figs. S1 to S18
Tables S1 to S5
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Supplementary Materials
Supplementary Text
Figs. S1 to S18
Tables S1 to S5