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Published in final edited form as: Phytochemistry. 2021 Jan 29;184:112672. doi: 10.1016/j.phytochem.2021.112672

A pair of threonines mark ent-kaurene synthases for phytohormone biosynthesis

Reid Brown a, Meirong Jia a, Reuben J Peters a,*
PMCID: PMC7990685  NIHMSID: NIHMS1665690  PMID: 33524857

Abstract

All land plants (embryophytes) must contain an ent-kaurene synthase (KS), as the ability to produce this olefin from ent-copalyl diphosphate (ent-CPP) is required for phytohormone biosynthesis. These KS have frequently given rise to other class I diterpene synthases that catalyze distinct reactions for more specialized plant metabolism. Indeed, the prevalence of such gene duplication and neofunctionalization has obscured phylogenetic assignment of function. Here a pair of threonines is found to be conserved in all land plant KS involved in phytohormone biosynthesis, and their role in enzyme function investigated. Surprisingly, these threonines are not required, nor even particularly important for efficient production of ent-kaurene from ent-CPP. In addition, these threonines do not seem to affect protein structure or stability. Moreover, the absence of codon bias and positioning within an intron do not support a role in transcription or translation either. Despite their lack of apparent function, this pair of threonines are nevertheless completely conserved in all embryophyte KS from phytohormone biosynthesis. Thus, regardless of exact role, this serves as a diagnostic mark for such KS, enabling more confident distinction of these critical enzymes.

Keywords: Gibberellins, terpene synthases, enzyme, evolution

1. Introduction

ent-Kaurene (1) is a key intermediate for phytohormone biosynthesis in all land plants (embryophytes), gibberellins (GA) in the case of vascular plants (tracheophytes) and other derived molecules in the earlier diverging lineages leading to the extant liverworts (marchantiophytes), mosses (bryophytes) and hornworts (anthocerotophytes)(Zi et al., 2014). Consistent with an early origin in embryophyte evolution, all land plants contain terpene synthases (TPSs) for the production of 1 (Chen et al., 2011). In particular, 1 is produced from the general precursor (E,E,E)-geranylgeranyl diphosphate via initial bicyclization to ent-CPP (2) catalyzed by a class II diterpene cyclase termed a CPP synthase (CPS), followed by further cyclization and rearrangement to 1 catalyzed by a class I diterpene synthase (KS)(Figure 1). The ancestral TPS seems to have been a bifunctional CPSKS, representing fusion of a CPS and KS, with γβα tridomain architecture stemming from the class I α domain and class II γβ didomain (Gao et al., 2012). Examples of CPSKS can still be found in nonseed plants, while seed plants (spermatophytes) contain separate CPS and KS resulting from an ancient gene duplication and subfunctionalization event (Zi et al., 2014).

Figure 1 |. KS reaction mechanism.

Figure 1 |

Notably, further gene duplication of the TPSs involved in production of 1 has given rise to a midsized gene family, wherein the vast majority have undergone neofunctionalization and are now involved in specialized (secondary) rather than phytohormone (primary) metabolism (Chen et al., 2011). Prominent among the resulting terpenoid natural products is the labdane-related diterpenoid superfamily, whose biosynthesis is characterized by the bicyclization of GGPP catalyzed by class II diterpene cyclases (Peters, 2010), with the resulting products almost invariably further cyclized and/or rearranged by subsequently acting class I diterpene synthases derived from KS, which are then often termed KS-like (KSL)(Zi et al., 2014). These KSL form a distinct TPS subfamily (TPS-e), which is distinguished not only by closer phylogenetic relationship to KS, but also retention of the ancestral γβα tridomain architecture, in contrast to the βα didomain architecture (reflecting an ancient γ domain loss event), exhibited by most other class I TPS (Chen et al., 2011).

Unfortunately, phylogenetic analysis does not clearly distinguish between the KS required for phytohormone biosynthesis and the KSL that have undergone diversion to secondary metabolism via neofunctionalization (Figure 2). Accordingly, KS serves as a genetic reservoir that has repeatedly been drawn upon to create the observed diversity of labdane-related diterpenoids from land plants, particularly the angiosperms (Jackson et al., 2014). However, this then confounds phylogenetic assignment of catalytic, as well as physiological function. Indeed, 1 not only serves as an intermediate in phytohormone biosynthesis (primary metabolism), but also as a precursor to specialized labdane-related diterpenoid natural products (secondary metabolism), particularly in the Poaceae plant family (Murphy and Zerbe, 2020), further confounding assignment of physiological function.

Figure 2 |. Representative phylogenetic tree for plant KS(L)/TPS-e subfamily.

Figure 2 |

Green lines indicate KS activity, red lines KSL that mediate alternative product outcome. Green text indicates KS with known or assumed role in phytohormone biosynthesis, while blue text indicates enzymes known to produce 1 for secondary metabolism – i.e., in maize (Fu et al., 2016).

More generally, class I TPSs catalyze lysis of the allylic diphosphate ester bond in isoprenyl diphosphate precursors (Christianson, 2006). For this purpose, all class I TPSs contain two characteristic motifs, DDxxD and NSE/DTE, which coordinate the requisite trio of divalent magnesium (Mg2+) co-factors (Aaron and Christianson, 2010). In addition to these two motifs, it has been shown that KSs contain an isoleucine that is important for the production of 1, with threonine substitution short-circuiting the complex reaction leading to 1, with deprotonation of the intermediate formed upon initial cyclization leading to production of the simpler ent-pimara-8(14),15-diene (Xu et al., 2007). Not surprisingly then, this isoleucine seems to be conserved throughout all embryophyte KSs (Jia and Peters, 2016; Zerbe et al., 2012), although it is present in many KSLs and can serve a similar role in enabling more complex reactions therein as well (Morrone et al., 2008). Here further sequence analysis was applied to the KS(L)/TPS-e subfamily, leading to discovery of a pair of threonines more specifically conserved in those KSs involved in phytohormone biosynthesis. Although extensive mutagenesis and biochemical characterization did not yield clear insight into the importance of these threonines, this pair nevertheless mark such KSs, providing additional means by which these important enzymes can be distinguished.

2. Results and Discussion

Previous work demonstrated key role of an isoleucine conserved in KSs from liverwort, lycophyte, gymnosperm, monocot and dicot, as well as bifunctional CPSKS from a moss (Jia and Peters, 2016; Zerbe et al., 2012). Intriguingly, this isoleucine is found in a PI(V/I) or PIx motif. While no structure has yet been reported for a member of the TPS-e subfamily, alignment with structurally defined TPS indicate that this motif is in the G1/2 helix wherein the proline seems likely to define the kink between the two subhelices. Notably, this helical break has been suggested to be a key component of substrate binding and catalysis in class I TPS (Baer et al., 2014), which might contribute to the hypothesized ability of substituted threonine at this position to act as a Brønsted-Lowry base (Jia et al., 2019). Regardless, given the importance of this isoleucine for the production of 1, the PIx motif is found in all TPSs that catalyze such product outcome, including not only those for phytohormone biosynthesis but also those involved in secondary metabolism as well.

Beyond the PIx motif it was noticed that the residues upstream of the DDxxD Mg2+-binding motif exhibit an intriguing conservation pattern. In particular, there is a pair of threonines found in all KSs involved in phytohormone biosynthesis, but not in many KSLs, leading to a more specific TTxxDDxxD motif in such KSs (Figure 3A & B). While the DDxxD motif is involved in binding the Mg2+ co-factors necessary for class I TPS catalysis, modeling indicates that the preceding KS-specific pair of threonines sit in the active site below the expected Mg2+ coordination sphere (Figure 3C). Notably, at least the first threonine does not seem to be required for production of 1, as this position contains an isoleucine in the maize (Zea mays) ZmKSL5, which specifically produces 1 but is involved in secondary rather than primary (phytohormone) metabolism (Fu et al., 2016). Indeed, ZmKSL5 provides an example of independent loss of the γ domain relative to both the ancient such event that occurred in the TPS family (Figure 2), as well as another example that also occurred in the TPS-e subfamily but within the dicot lineage instead (Hillwig et al., 2011), consistent with extensive drift away from the KS required for gibberellin phytohormone biosynthesis.

FIGURE 3 |. Identification of the TT motif.

FIGURE 3 |

A) Representative alignment of KSs spanning plant evolution with the KS specific pair of threonines (TT motif) indicated by asterisks (*) above the alignment. B) Sequence logos demonstrating the absolute conservation of this TT motif in KS (bottom) relative to the derived KS(L)/TPS-e subfamily more generally (top). C) Location of TT motif in active site from model of AtKS (blue) containing 2-fluoroGGPP (green) and Mg2+ co-factors (magenta) derived from the template co-crystal structure of taxadiene synthase (Koksal et al., 2011).

To investigate the importance of this KS-specific pair of threonines for production of 1, extensive mutagenesis was carried out with the model KS from Arabidopsis thaliana (AtKS)(Yamaguchi et al., 1998), which has been proven to be amenable to mutational analysis (Jia et al., 2017; Xu et al., 2007). Specifically, each of these threonines was replaced with one of the alternative residues found in other TPS-e subfamily members, which included valine, leucine, isoleucine, methionine, alanine and phenylalanine. This led to creation of multiple single mutants at both the T527 and T528 positions. In addition, given the approximately isosteric nature of valine and threonine, as well as prevalence of valine as the alternative residue at least at the first position (Figure 3B), a T527V/T528V (TT/VV) double mutant also was constructed. The impact of these mutations on production of 1 was analyzed via incorporation of the mutant, as well as wild-type (WT) AtKS into a modular metabolic engineering system wherein Escherichia coli is induced to produce the relevant substrate 2 (Cyr et al., 2007). Strikingly, while all mutants except for T527F were active, these still exclusively produced 1 (Figure 4). Thus, although substitution with a bulky aromatic residue at the first position appears to disrupt either protein folding and/or catalysis, this pair of threonines is clearly not required for selective production of 1.

FIGURE 4 |. TT motif is not required for the production of ent-kaurene (1).

FIGURE 4 |

Extracted ion (m/z = 272) chromatograms demonstrating the production of 1 by all mutants (as indicated) by comparison to wild-type (WT) AtKS.

To investigate the importance of this pair of threonines for catalytic efficiency (kcat/KM), steady-state kinetic analysis was carried out with the single and double valine mutants, as well as WT AtKS (Figure 5). Only small (<2-fold) differences were noted with the T527V and T528V single mutants for the resulting catalytic rate (kcat) and Michaelis pseudo-binding constant (KM), with no significant difference in catalytic efficiency (Table 1). Even with the TT/VV double mutant, the somewhat more significant 4-fold reduction in kcat is offset by a ~5-fold improvement in KM, such that its catalytic efficiency is, if anything, slightly higher than WT. Hence, this pair of threonines do not have a critical role in catalytic efficiency.

FIGURE 5 |. TT motif has minimal effect on catalytic efficiency.

FIGURE 5 |

Curve fit of Michaelis-Menton equation to data for wild-type (WT) and indicated mutants of AtKS.

Table 1|.

Kinetic Constants

AtKS kcat (s−1) KM (μM) kcat/KM (s−1 M−1)
WT 1.3 ± 0.2 8 ± 3 1.7 ×104
T527V 1.7 ± 0.2 9 ± 2 1.8 ×104
T528V 0.9 ± 0.1 5 ± 2 1.9 ×104
TT/VV 0.33 ± 0.02 1.5 ± 0.5 2.2 ×104

To investigate the impact of this pair of threonines for protein structure, circular dichroism spectra were measured for WT versus the TT/VV double mutant (Figure 6). Consistent with the observed catalytic efficiencies, the equivalent shapes of these spectra indicate that even the TT/VV double mutant retains the same amount of secondary structure as WT AtKS. In addition, subjecting these to a thermal shift assay also did not reveal any significant difference in overall stability, with derivative melting temperatures of 60.4 ± 0.5 °C for WT versus 60.1 ± 0.5 °C for the TT/VV double mutant. Accordingly, this pair of threonines do not seem to have an important role in protein structure or stability.

FIGURE 6 |. TT motif has minimal effect on protein structure.

FIGURE 6 |

CD spectra for wild-type (WT) and the T527V/T528V (TT/VV) double mutant of AtKS (note that the minimal differences observed here are not reproducible).

Given the lack of evidence of a critical role for this pair of threonines at the enzymatic structure-function level, the encoding nucleotide sequence was considered. However, the relevant codons did not exhibit any consistent conservation pattern, nor were these located near an exon boundary (Figure 7). Thus, it does not seem likely that the pair of threonines are conserved for any role in transcription and/or translation.

FIGURE 7 |. TT motif codons are not conserved.

FIGURE 7 |

A) Exon sizes from KS with known genomic sequence. B) Sequence of TT motif containing exon from AtKS (codons for TT motif in larger blue text). C) Sequence logo indicating lack of conservation of the codons for the TT motif – i.e., in the variable third/’wobble’ position.

Indeed, given the variation observed in the third/’wobble’ position it is clear that synonymous, although obviously not non-synonymous, changes are allowed for these codons in the KSs involved in phytohormone biosynthesis (i.e., dS > 0 while dN = 0). Accordingly, despite the lack of any important functional role found here, it is the amino acid residues (pair of threonines) that are under strict purifying selective pressure (i.e., dN/dS = 0). Given the variation observed in the rest of the TPS family at these positions, this selective pressure is clearly relived in other TPSs, including the KSLs from the TPS-e subfamily.

3. Conclusions

KSs play a key role in phytohormone biosynthesis and have been conserved throughout all land plants. Nevertheless, these are not readily recognizable as they have undergone repeated gene duplication and neofunctionalization to instigate secondary metabolism in in many of the major embryophyte lineages. Thus, the resulting TPS-e subfamily members are often most closely related by descent/lineage rather than physiological function. While an isoleucine from a PIx motif is known to be important for the production of 1, the key role this plays also means this is conserved in all such TPSs, including those involved in secondary rather than primary (phytohormone) metabolism. Here a pair of threonines was identified that is more specifically conserved in the KSs for phytohormone biosynthesis, found just upstream of the usual aspartate-rich Mg2+-binding motif, which is then TTxxDDxxD. Although the extensive mutational studies reported here failed to uncover a role for this pair of threonines in KS enzymatic structure-function, and nor is there any obvious role for the relevant codons in transcription or translation, its conservation throughout land plant evolution is striking. Indeed, this pair of threonines appears to be under strict purifying selective pressure and provides a mark for KS involved in phytohormone biosynthesis, which should prove useful in future bioinformatic-directed functional investigations of terpenoid metabolism, particularly given the increasing amount of sequence information available for plants.

4. Experimental

4.1. General

Unless otherwise noted, chemicals were purchased from Fischer Scientific and molecular biology reagents from Invitrogen. For the studies described here, a previously described pseudo-mature AtKS construct (Xu et al., 2007) was cloned into pET-100/d-TOPO via directional topoisomerization. This wild-type construct was then subjected to site-directed mutagenesis via whole-plasmid PCR using the primers in Supplemental Table 1. All mutants were verified by complete gene sequencing. All expression was carried out in the OverExpress C41 strain of E. coli (Lucigen).

4.2. Metabolic engineering

For analysis of product outcome each AtKS construct was co-transformed with the previously described pGGeC that leads to production of 2 (Cyr et al., 2007). The resulting recombinant strains were grown in 50 mL of TB media with the appropriate antibiotics (chloramphenicol and ampicillin) in 250 ml glass Erlenmeyer flasks at 37 °C to an OD600 of ~0.6. The cultures were then induced with 1 mM IPTG, the temperature reduced to 16 °C and allowed to grow for 72 hrs. before being extracted with 50 mL hexanes. For analysis by gas chromatography with mass spectral detection (GC-MS), 10 mL of each hexane extract were dried under N2 and resuspended in 0.1 mL hexanes.

4.3. Product analysis

GC-MS analysis was carried out with a Varian 3900 GC with a Saturn 2100T ion trap mass spectrometer in electron ionization (70 eV) mode over an Agilent HP-5MS column (Agilent, 19091S-433) with 1.2 mL/min helium flow rate. Samples (1 μL) were injected by a 8400 autosampler, with the injector port pre-heated to 250 °C and run in splitless mode. The column was held at 50 °C for three minutes, then heated at 15 °C/min to 300 °C, where the temperature was held for three minutes. Mass spectra were recorded as mass-to-charge ratios in the range of 90 to 650, beginning 13 min after injection to allow for the solvent front to pass. Products were identified by comparison of both retention time and mass spectra to the known production of 1 by wild-type AtKS. Samples were analyzed using the Varian WorkStation Software package (v6.9) and graphs produced using Igor Pro (v8.0.4.2).

4.4. Protein purification

AtKS was expressed and induced as described above, but in volumes of 3 L grown for only 18 hrs post-induction. Cell were harvested via centrifugation (Beckman Coulter JS 4.2 Series) at 4000 × g for 1 hr in 1 L centrifuge bottles. The resulting cell pellets were resuspended in buffer A (50 mM MOPSO, pH 6.8, 300 mM NaCl, 10% glycerol and 10 mM imidazole) and lysed via three rounds of homogenization (Emulsiflex-C3; 10,000–15,000 PSI) with two minutes of rest on ice in between each round. The crude lysate was clarified via centrifugation (Beckman Coulter Avant J-E) at 16,000 × g for 1 hour. Recombinant AtKS was then purified over 2 mL of HisPur Ni-NTA Resin using a Bio-Rad LP chromatography system run with LP Data View software (v1.01). Following loading of the clarified lysate the column was washed with 20 mL of buffer A and then eluted with buffer B (buffer A containing 250 mM imidazole). Fractions containing the protein were then dialyzed in 12–14 kDa MW cut-off Millipore dialysis tubing against a minimal buffer (MOPSO, 10% glycerol DTT, pH 6.8).

4.5. Kinetic analysis

Kinetic assays were carried out much as previously described (Jia et al., 2018). Briefly, radiolabeled ent-CPP substrate (2) was generated using tritium-labeled GGPP (American Radiochemicals) using the maize ent-CPP synthase An2/ZmCPS2 (Harris et al., 2005), purified as described above for AtKS. This initial reaction was carried out in class II assay buffer (50 mM HEPES, pH 7.2, 100 mM KCl, 0.1 mM MgCl2, 10% glycerol and 5 mM DTT), with 200 μM GGPP and 3 μM ZmCPS2. Reactions were performed in 1 mL volumes in 1.5 mL screw-top glass vials, which were incubated overnight at 30 °C in a hot water bath. Efficient substrate conversion (>95%) was verified via a parallel reaction run with non-radiolabeled GGPP, with subsequent dephosphorylation using calf intestinal alkaline phosphatase (Promega) to enable GC-MS analysis (run as described above). AtKS assays were run in triplicate in class I assay buffer (50 mM HEPES, pH 7.2, 7.5 mM MgCl2, 100 mM KCl, 10% glycerol, and 5 mM DTT) with 100 nM enzyme. These were equilibrated at 30 °C for 15 min prior to initiation of the reaction via the addition of substrate. These assays were run for times optimized by an initial time course to fall within the linear response range (5 min. for wild-type, 7.5 min for the single mutants and 10 min. for the double mutant). The reactions were stopped by addition of concentrated KOH and EDTA solution to bring the assay concentration to 0.2 M KOH and 15 mM EDTA, respectively, with rapid mixing via vortexing for 5 seconds. Products were extracted thrice with 1 mL hexanes, with the organic extract then passed over silica gel columns with a cap of anhydrous magnesium sulfate that had been pre-equilibrated with 1 mL hexanes, and which was further flushed with 1 mL hexanes after the three rounds of sample addition were finished. These pooled extracts were then combined with 5 mL BD Scintillation fluid in Scintillation vials and vortexed vigorously. Samples were left for 1 hour to equilibrate and then counted with a Perkin Elmer Tri-Carb 4910TR Liquid Scintillation Analyzer. The raw counts presented in the QuantaSmart software (v5.1) were then normalized to a standard curve generated from samples with known radiolabeled substrate concentrations. Finally, kinetics parameters were determined with MATLAB (R2019b, update 4 v9.7.0). Figure 4 was generated with GraphPad Prism 8.

4.5. Structural analysis

Circular dichroism was measured using a Biologic MOS-500 unit connected to an SFM 400 Cuvette mount held at 22 degrees Celsius using an ISOTEMP control water circulating unit. Samples consisted of 400 μL of 1 mg/mL fresh protein, purified as described above, which was loaded into the 10 mm Starna Cells Quartz Spectrophotometer cell. Spectra were obtained at 1 nm wavelength increments measured for 0.25 sec and averaged over three replicates using the Biokine software (v4.73).

4.6. Protein stability analysis

A thermal shift assay to measure potential differences in melting temperature was performed with fresh protein, purified as described above, with a OneStepPlus qPCR machine located in the Iowa State DNA facility. Samples were loaded in eight replicate wells on a Microtemp Fast Optical 96-well Reaction Plate and normalized to buffer controls loaded in three replicate wells. A total volume of 20 μL was assayed with 1x SYPRO Orange dye and a final concentration of 1 mg/mL protein. The results were analyzed with the Applied Biosystems Protein Thermal Shift Software (v1.1).

4.7. Bioinformatic analyses

Sequence analyses were carried out with CLC Main Workbench (version 20.0.4). The analyzed KS(L)/TPS-e subfamily members are listed in Supplemental Table 2. While the bifunctional CPSKS are relevant here, these fall within the TPS-c subfamily (Chen et al., 2011), which allowed their use as the designated outgroup for the phylogenetic tree, which was prepared using FigTree (v1.4.4). Sequence logo figures were created using the Berkeley WebLogo tool (Crooks et al., 2004). The AtKS model was generated via the Swiss-Model webserver, using as template the structure of taxadiene synthase with 2-fluoroGGPP and Mg2+ (Koksal et al., 2011). To provide context for the relative position of the pair of threonines investigated here the 2-fluoro-GGPP and Mg2+ from the template (PDB: 3P5R) were then added back to the AtKS model. Finally, exons for the selected genes were obtained using The Arabidopsis Internet Resource for AtKS and the US National Library of Medicines Nucleotide Blast utility to find the others presented in Figure 7, which was organized using Snapgene (v5.0.7).

Supplementary Material

1

Highlights.

  • Identification of pair of threonines marking ent-kaurene synthases (KSs)

  • Striking conservation among all land plants

  • Threonines not required for production of ent-kaurene

  • Nevertheless, present in all KSs involved in phytohormone biosynthesis

Acknowledgements

This study was supported by a grant from the NIH (GM131885) to R.J.P.

Footnotes

Declaration of interests

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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