Limonene Synthase

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You can go back to main page of the kinetic model here.

Model construction progress :

Enzyme Pathway Keq Kcat VmaxF KmGPP KmSub2 [GPP] [Sub2] VmaxR KmLim KmPP [Limonene] [PP]
02_LimSynth Limonene Biosynthesis 1 5 3 >6 no Sub2 4 no Sub2 estimated estimated estimated estimated estimated

Legend:

Have not started ·· 1 -2 data found ·· 3-4 data found ·· sufficient data found/estimated ·· data distribution generated ·· data sampled
to do DONE!


What we know

Issues

Strategies

Reaction catalysed

geranyl diphosphate \rightleftharpoons (−)-(4S)-limonene + diphosphate

Metabolite Background Information

Long metabolite names are abbreviated in the model for clarity and standard identification purposes.

Metabolite Abbreviation Chemical Formula Molar mass (g/mol) ChEBI ChEMBL PubChem
geranyl diphosphate GPP C10H20O7P2 314.209 17211 41432 445995
(-)-4S-limonene Limonene C10H16 136.24 15384 449062 22311 or 439250
diphosphate PP O7P2 173.94 644102
limonene synthase LimSynth 72.4 kDa [1] ; 60kDa [2]

Equation Rate

The reversible Michaelis-Menten equation to model the dynamic changes of LimSynth is:

V_\mathrm{LimSynth} = Vmax_\mathrm{forward} * \cfrac {\cfrac{[GPP]}{Km_\mathrm{GPP}} * \left ( 1 - \cfrac {[Limonene]*[PP]}{[GPP]*K_\mathrm{eq}} \right )}{1 + \cfrac {[GPP]}{Km_\mathrm{GPP}} + \cfrac {[Limonene]}{Km_\mathrm{Limonene}} + \cfrac {[PP]}{Km_\mathrm{PP}} + \cfrac {[Limonene]*[PP]}{Km_\mathrm{Limonene}*Km_\mathrm{PP}}}

where :

Parameter Description Units
VLimSynth Reaction rate for Limonene Synthase
Vmaxforward Maximum reaction rate towards the production of limonene ref
KmGPP Michaelis-Menten constant for GPP mM
KmLimonene Michaelis-Menten constant for Limonene mM
KmPP Michaelis-Menten constant for PP mM
Keq Equilibrium constant
[GPP] GPP concentration mM
[Limonene] Limonene concentration mM
[PP] PP concentration mM

Strategies for estimating the kinetic parameter values

Calculating the Equilibrium Constant

Standard Gibbs Free energy

Standard Gibbs Free energy for Limonene Synthase from MetaCyc (EC 4.2.3.16) is -28.049988 kcal/mol [3].

SI derived unit for Gibbs free energy is Joules per mol (J mol-1). 1 kJ·mol−1 is equal to 0.239 kcal·mol−1.

Therefore, the Gibbs free energy for Limonene synthase in kJ mol-1 is:

Failed to parse (Cannot store math image on filesystem.): \cfrac {1}{0.239 kcal.mol^-1} * -28.049988 kcal.mol^-1
Failed to parse (Cannot store math image on filesystem.): = -117.36396 kJmol^-1

The Equilibrium Constant

The equilibrium constant can be calculated using the Van't Hoff Isotherm equation:

K_\mathrm{eq} = exp \left ( \cfrac {-ΔG^{°'}}{RT} \right )


Failed to parse (Cannot store math image on filesystem.): = exp \left ( \cfrac {-(- 117.36396 \text { kJmol}^{-1})}{ (8.31 \text{ JK}^{-1} \text { mol}^{-1} * 289 K} \right )

Failed to parse (Cannot store math image on filesystem.): = exp \left ( \cfrac { + 117.36396 \text { kJmol}^{-1} }{ 2401.59 \text{ JK}^{-1}\text { mol}^{-1} }\right)

Failed to parse (Cannot store math image on filesystem.): = exp \left ( \cfrac{ 117.364 * 10^3 \text { Jmol}^{-1}}{2401.59 \text{ JK}^{-1}\text { mol}^{-1}} \right)

Failed to parse (Cannot store math image on filesystem.): =exp \left ( 48.8693 \right )

Failed to parse (Cannot store math image on filesystem.): = 1.6736 * 10^{21}


where;

Keq Equilibrium constant
-ΔG° Gibbs free energy change. For Limonene Synthase it is -117.364 kJmol-1
R Gas constant with a value of 8.31 JK-1mol-1
T Temperature which is always expressed in Kelvin

Kinetic Parameter Values

Values for the kinetic parameter required to simulate this model can be obtained from published and unpublished literature.

A more detailed descriptions of the values listed above can be found HERE , where I've linked and highlighted where these data came from.

Substrate, Product & Enzyme Concentration Values

Concentration Unit Substrate / Product Directionality Organism Method notes References
25 µM GPP forward E. coli [4]
10 µM GPP forward Cannabis sativa L. [5]
10 µM GPP forward Mentha sp. [6]
2 µM GPP forward Citrus limon [7]
150 µg Limonene synthase - E. coli [4]
0.07 µM Limonene synthase - Citrus sinensis with 200µM GPP as substrate, at 20°C, pH7.5 [8]
0.26 µM Limonene synthase - Citrus sinensis with 200µM GPP, at 37°C, pH7.5 [8]
200 µM GPP forward Citrus sinensis used for Limonene synthase activity assay at 20°C and 37°C, pH 7.5 [8]
100 µM GPP - Citrus limon [7]
0.1 µM GPP - Citrus limon [7]

Km Values

Km (mM) Unit Substrate / Product Directionality Organism Method notes References
0.13 mM GPP forward Citrus sinensis Enzyme assay, pH 7.5, 20°C [8]
0.00125 mM GPP forward Ricciocarpos natans [9]
0.0018 mM GPP forward Mentha piperita Ref
0.01176 ± 0.00245 mM GPP forward Cannabis sativa L. [10]
0.0031 mM GPP forward Citrus limon ref
0.016 mM GPP forward Escherichia coli [4]
0.0067 mM GPP forward Escherichia coli (wt) [11]
0.0068 mM GPP forward Cannabis sativa L. enzyme assay, pH7.5, 40°C [5]
0.0086 mM GPP forward Escherichia coli (Q53). [4]
0.006 mM GPP forward Escherichia coli (E57). [4]
0.0126 mM GPP forward Escherichia coli (R58). [4]
0.0299 mM GPP forward Escherichia coli (R59). [4]
0.0324 mM GPP forward Escherichia coli (R58P59) [4]
0.0275 mM GPP forward Escherichia coli (R58A59) [4]
0.0188 mM GPP forward Escherichia coli (S60) [4]
0.0007 mM GPP forward Citrus limon [7]
0.00625 ± 0.00041 mM GPP forward Cannabis sativa L [10]

Vmax values

Vmax Unit Directionality Organism Organism References
0.08 µmol/min/mg forward Cannabis sativa L. [10]
0.12 ± 0.01 µmol/min/mg forward Cannabis sativa [10]
0.4748 µmol/min/mg forward Citrus limon References
0.53 µM/min forward Citrus sinensis 200µM GPP, 20°C, [E]: 0.07µM, pH7.5 [8]
0.64 µM/min forward Citrus sinensis 200µM GPP, 37°C, [E]: 0.26µM, pH7.5 [8]
Vmax Unit Directionality Organism References

Kcat values

Kcat Unit Directionality Organism Reference
0.13 s-1 forward Citrus sinensis [8]
0.04 s-1 forward Citrus sinensis [8]
0.3 s-1 forward Mentha piperita & Mentha spicata [6]
0.09 s-1 forward Cannabis sativa L. [10]
0.13 s-1 forward Cannabis sativa L. [10]
0.02 s-1 forward E. coli [4]
0.082 s-1 forward Cannabis sativa L. [5]
0.0024 s-1 forward E. coli (wt) [11]
0.0020 s-1 forward E. coli (preprotein) [4]
0.0036 s-1 forward E. coli (Q54) [11] [4]
0.0034 s-1 forward E. coli (E57) [11] [4]
0.0037 s-1 forward E. coli (R58) [11] [4]
<0.0004 s-1 forward E. coli (R59) [11] [4]
<0.0004 s-1 forward E. coli (R58P59) [11] [4]
<0.0004 s-1 forward E. coli (R58A59) [11] [4]
<0.0004 s-1 forward E. coli (S60) [11] [4]

Extracting Information from Limonene Production Rates

The production rates would reflect on the flux for this enzyme, and this would provide provide the insights on the Vmax of this enzyme.

Amount produced (mg/L) Time (H) Organism Description Reaction Flux (µM/s)
5 24 Escherichia coli Possible reason for the low limonene production might due to the insufficient supply of IPP and DMAPP [12]. 0.0255
335 48 Escherichia coli Engineered E.coli in which heterologous MVA pathway was installed [13]. 0.8537
35.8 48 Escherichia coli E.coli was engineered to express GPPS, LS, DXS, and IDI [14] . 0.0912
4.87 48 Escherichia coli This was the initial titer. The study established a limonene biosynthesis pathway in E.coli using four different polycistronic operons based on 3 vectors with varied expression strength [14]. 0.0124
17.4 48 Escherichia coli Using a plasmid with DXS and IDI over expressed [14]. 0.0445
430 72 Escherichia coli [13] 0.7306

Parameter estimation

This section can be found HERE

Simulations

Simulations performed can be found HERE.

References

  1. ↑ Turner,G. et. al.1999. "Limonene synthase, the enzyme responsible for monoterpene biosynthesis in peppermint, is localized to leucoplasts of oil gland secretory cells", Plant Physiology 120(3): 879-886
  2. ↑ Maruyama, T. et. al. 2002. "Molecular cloning, functional expression and characterization of d-Limonene synthase from Agastache rugosa" Biol. Pharm. Bull. 25(5): 661-665
  3. ↑ Latendresse M. (2013). "Computing Gibbs Free Energy of Compounds and Reactions in MetaCyc."
  4. ↑ 4.00 4.01 4.02 4.03 4.04 4.05 4.06 4.07 4.08 4.09 4.10 4.11 4.12 4.13 4.14 4.15 4.16 4.17 4.18 Williams, D.C. et. al. 1998. "Truncation of Limonene Synthase preprotein provides a fully active 'pseudomature' form of this monoterpene cyclase and reveals the function of the amino-terminal arginine pair ",Biochemistry, 37:12213-12220 Cite error: Invalid <ref> tag; name "Williams1998" defined multiple times with different content
  5. ↑ 5.0 5.1 5.2 Gunnewich, N., et al. 2006."Functional expression and characterization of trichome-specific (-)-limonene synthase and (+)-α-pinene synthase from Cannabis sativa", Natural Product Communications, 0(0): pp1-10 Cite error: Invalid <ref> tag; name "Gunnewich2006" defined multiple times with different content
  6. ↑ 6.0 6.1 Alonso et. al. 1992. "Purification of 4S-Limonene Synthase, a Monoterpene Cyclase from the Glandular Trichomes of Peppermint (Mentha x piperita) and Spearmint (Mentha spicata)", The Journal of Biological Chemistry, 267(11):7582-7587
  7. ↑ 7.0 7.1 7.2 7.3 Lücker, J. et. al. 2002."Monoterpene biosynthesis in lemon (Citrus limon). cDNA isolation and functional analysis of four monoterpene synthases", Eur. J. Biochem. 269: pp3160-3171
  8. ↑ 8.0 8.1 8.2 8.3 8.4 8.5 8.6 8.7 Entova, S. 2013. "Kinetic characterization, crystallization, and photosynthetic expression of (+)-4R-limonene synthase from C. sinensis", Masters Thesis, Brandeis Univeristy Cite error: Invalid <ref> tag; name "Entova2013" defined multiple times with different content
  9. ↑ Adam, K. et. al. 1996. "Partial purification and characterization of a monoterpene cyclase, limonene synthase, from the liverwort Ricciocarpos natans. 332(2):pp 352-356.
  10. ↑ 10.0 10.1 10.2 10.3 10.4 10.5 Gunnewich, N. 2008. "Expression and characterization of terpene synthases from Cannabis sativa L. and Salvia sclarea L. Doctoral thesis. Cite error: Invalid <ref> tag; name "Gunnewich2008" defined multiple times with different content
  11. ↑ 11.0 11.1 11.2 11.3 11.4 11.5 11.6 11.7 11.8 Ogura, K. & Koyama, T. 1997, "Dynamic aspects of Natural Products Chemistry". pp 1-23.
  12. ↑ Carter, Ora A. et. al.2013. "Monoterpene biosynthesis pathway construction in Escherichia coli",Phytochemistry, 64:425–433, 2003.
  13. ↑ 13.0 13.1 Alonso-Gutierez et. al. 2013. "Metabolic engineering of Escherichia coli for limonene and perillyl alcohol production", Metabolic Engineering, 19:33-41 Cite error: Invalid <ref> tag; name "AlonsoGutierez2013" defined multiple times with different content
  14. ↑ 14.0 14.1 14.2 Du et. al. 2014. "Enhanced limonene production by optimizing the expression of limonene biosynthesis and MEP pathway genes in E.coli", Bioprocessing and Bioprocessing, 1:10
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