Difference between revisions of "Limonene Synthase"

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(→‎Kcat values)
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==== Kcat values ====
 
  
 
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{| class="wikitable sortable"
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! Parameter || Direction || Substrate || Value || Unit || Method || M_w || Organism || O_w || Expression_vector || E_w || Enzyme || En_w || pH || P_w || Temperature || T_w || Total_weight || Description || Reference
 
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! style="border: 1px solid black; padding: 5px; background: #ffdead; width: 50px;" | Kcat
+
| Kcat || Forward || GPP || 0.0004 || 1/s || in vitro (+2) || 2 || Unrelated (+0) || 0 || E.coli (+4) || 4 || Identical (+4) || 4 || 7.0 (+4) || 4 || different temperature (+0) || 0 || 128 || Protein from Mentha sp, expressed in E.coli and truncated (R59), pH7.0, 1-50 mg [E], 25 µM [GPP], limonene produced 94%, 2% myrcene, pinene  || Williams1998
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! style="border: 1px solid black; padding: 5px; background: #ffdead;" | Unit
 
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! style="border: 1px solid black; padding: 5px; background: #ffdead;" | Directionality
 
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! style="border: 1px solid black; padding: 5px; background: #ffdead;" | Organism
 
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! style="border: 1px solid black; padding: 5px; background: #ffdead; width: 300px;" | Method notes
 
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! style="border: 1px solid black; padding: 5px; background: #ffdead;" | Reference
 
 
|-
 
|-
−
| 0.13
+
| Kcat || Forward || GPP || 0.0004 || 1/s || in vitro (+2) || 2 || Unrelated (+0) || 0 || E.coli (+4) || 4 || Identical (+4) || 4 || 7.0 (+4) || 4 || different temperature (+0) || 0 || 128 || Protein from Mentha sp. expressed in E.coli , truncated R58P59, pH7.0, 1-50 mg [E], 25 µM [GPP], limonene produced 94%, 2% myrcene, pinene  || Williams1998
−
| s<sup>-1</sup>
 
−
| forward
 
−
| ''Citrus sinensis''
 
−
| 200mM GPP, 20°C, [E]: 0.07 mM, pH7.5
 
−
| <ref name="Entova2013"></ref>
 
 
|-
 
|-
−
| 0.04
+
| Kcat || Forward || GPP || 0.0004 || 1/s || in vitro (+2) || 2 || Unrelated (+0) || 0 || E.coli (+4) || 4 || Identical (+4) || 4 || 7.0 (+4) || 4 || different temperature (+0) || 0 || 128 || Protein from Mentha sp. expressed in E.coli , truncated R58A59, pH7.0, 1-50 mg [E], 25 µM [GPP], limonene produced 94%, 2% myrcene, pinene  || Williams1998
−
| s<sup>-1</sup>
 
−
| forward
 
−
| ''Citrus sinensis''
 
−
| 200mM GPP, 37°C, [E]: 0.26mM, pH7.5
 
−
| <ref name="Entova2013"></ref>
 
 
|-
 
|-
−
| 0.3
+
| Kcat || Forward || GPP || 0.0004 || 1/s || in vitro (+2) || 2 || Unrelated (+0) || 0 || E.coli (+4) || 4 || Identical (+4) || 4 || 7.0 (+4) || 4 || different temperature (+0) || 0 || 128 || Protein from Mentha sp. expressed in E.coli , S60, pH7.0, 1-50 mg [E], 25 µM [GPP], limonene produced 94%, 2% myrcene, pinene  || Williams1998
−
| s<sup>-1</sup>
 
−
| forward
 
−
| ''Mentha piperita'' & ''Mentha spicata''
 
−
| pH7.0, 30°C, Limonene synthase assay, 10 μM [GPP], 1-10 μg protein per mixture
 
−
| <ref name="Alonso1992">Alonso et. al. 1992. [http://www.ncbi.nlm.nih.gov/pubmed/1559995 "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]</ref>
 
 
|-
 
|-
−
| 0.09
+
| Kcat || Forward || GPP || 0.002 || 1/s || in vitro (+2) || 2 || Unrelated (+0) || 0 || E.coli (+4) || 4 || Identical (+4) || 4 || 7.0 (+4) || 4 || different temperature (+0) || 0 || 128 || Protein from Mentha sp. expressed in E.coli preprotein, pH7.0, 1-50 mg [E], 25 µM [GPP], limonene produced 94%, 2% myrcene, pinene  || Williams1998
−
| s<sup>-1</sup>
 
−
| forward
 
−
| ''Cannabis sativa '' L.
 
−
|
 
−
| <ref name="Gunnewich2008"></ref>
 
 
|-
 
|-
−
| 0.13
+
| Kcat || Forward || GPP || 0.0024 || 1/s || in vitro (+2) || 2 || Unrelated (+0) || 0 || E.coli (+4) || 4 || Identical (+4) || 4 || 7.0 (+4) || 4 || different temperature (+0) || 0 || 128 || Protein from Mentha sp. expressed in E.coli wt, pH7.0, 1-50 mg [E], 25 µM [GPP], limonene produced 94%, 2% myrcene, pinene  || Williams1998
−
| s<sup>-1</sup>
 
−
| forward
 
−
| ''Cannabis sativa L.''
 
−
|  
 
−
| <ref name="Gunnewich2008"></ref>
 
 
|-
 
|-
−
| 0.02
+
| Kcat || Forward || GPP || 0.0034 || 1/s || in vitro (+2) || 2 || Unrelated (+0) || 0 || E.coli (+4) || 4 || Identical (+4) || 4 || 7.0 (+4) || 4 || different temperature (+0) || 0 || 128 || Protein from Mentha sp. expressed in E.coli E57, pH7.0, 1-50 mg [E], 25 µM [GPP], limonene produced 94%, 2% myrcene, pinene  || Williams1998
−
| s<sup>-1</sup>
 
−
| forward
 
−
| ''E. coli''
 
−
|
 
−
| <ref name= "Williams1998"></ref>
 
 
|-
 
|-
−
| 0.082
+
| Kcat || Forward || GPP || 0.0036 || 1/s || in vitro  || 2 || Unrelated (+0) || 0 || E.coli (+4) || 4 || Identical (+4) || 4 || 7.0 (+4) || 4 || different temperature (+0) || 0 || 128 || Protein from Mentha sp. expressed in E.coli Q54, pH7.0, 1-50 mg [E], 25 µM [GPP], limonene produced 94%, 2% myrcene, pinene  || Williams1998
−
| s<sup>-1</sup>
 
−
| forward
 
−
| ''Cannabis sativa L.''
 
−
|CsTPS1, pH6.5, 40°C, 1.25 mg [LimSynth] per 500ml assay mixture, 10mM [GPP]
 
−
| <ref name="Gunnewich2006"> Günnewich et. al. 2006. [https://www.researchgate.net/publication/42089356 "Functional expression and characterization of trichome-specific (-)-limonene synthase and (+)-α-pinene synthase from ''Cannabis sativa''", Natural Product Communications, 0(0):1-10]</ref>
 
 
|-
 
|-
−
| 0.000039
+
| Kcat || Forward || GPP || 0.0037 || 1/s || in vitro  || 2 || Unrelated (+0) || 0 || E.coli  || 4 || Identical (+4) || 4 || 7.0 (+4) || 4 || different temperature (+0) || 0 || 128 || Protein from Mentha sp. expressed in E.coli R58, pH7.0, 1-50 mg [E], 25 µM [GPP], limonene produced 94%, 2% myrcene, pinene  || Williams1998
−
| s<sup>-1</sup>
 
−
| forward
 
−
| ''Citrus sinenesis''
 
−
| Limonene synthase assay, with 40 μM GPP, 37°C
 
−
| <ref name="Olsen2011"> Olsen, S. N. 2011. [http://bir.brandeis.edu/handle/10192/24424?show=full] "Isolation, purification, and characterization of (+)-4R-limonene synthase: The first step in exploring enzyme stereospecificity in terpenoid biosynthesis" Masters Thesis, Brandeis University, </ref>
 
 
|-
 
|-
−
| 0.0024
+
| Kcat || Forward || GPP || 0.02 || 1/s || in vitro  || 2 || Unrelated || 0 || E.coli || 4 || Identical (+4) || 4 || 7.0 (+4) || 4 || different temperature (+0) || 0 || 128 || Protein from Mentha sp. expressed in E.coli recombinant preprotein , pH7.0, 1-50 mg [E], 25 µM [GPP], limonene produced 94%, 2% myrcene, pinene  || Williams1998
−
| s<sup>-1</sup>
 
−
| forward
 
−
| ''E. coli'' (wt)
 
−
|
 
−
| <ref name= "Ogura1997"></ref>
 
 
|-
 
|-
−
| 0.0020
+
| Kcat || Forward || GPP || 0.04 || 1/s || in vitro  || 2 || Unrelated  || 0 || E.coli || 4 || Identical (+4) || 4 || close range (+2) || 2 || 37°C (+4) || 4 || 256 || Citrus sinensis, expressed in E.coli 200µM GPP, 37°C, [E]: 0.26µM, pH7.5 || Entova2013
−
| s<sup>-1</sup>
 
−
| forward
 
−
| ''E. coli'' (preprotein)
 
−
|
 
−
| <ref name= "Williams1998"></ref>
 
 
|-
 
|-
−
| 0.0036
+
| Kcat || Forward || GPP || 0.082 || 1/s || in vitro  || 2 || Unrelated  || 0 || E.coli || 4 || Identical (+4) || 4 || close range (+2) || 2 || 36°C - 40°C (+2) || 2 || 128 || Cannabis sativa L., expressed in E.coli,CsTPS1, pH6.5, 40°C, 1.25 mg [LimSynth] per 500ml assay mixture, 10mM [GPP] || Gunnewich2008
−
| s<sup>-1</sup>
 
−
| forward
 
−
| ''E. coli'' (Q54)
 
−
|
 
−
| <ref name= "Ogura1997"></ref> <ref name="Williams1998"></ref>
 
 
|-
 
|-
−
| 0.0034
+
| Kcat || Forward || GPP || 0.09 || 1/s || in vitro  || 2 || Unrelated  || 0 || E.coli || 4 || Identical (+4) || 4 || close range (+2) || 2 || 36°C - 40°C (+2) || 2 || 128 || Cannabis sativa L., expressed in E.coli,Km of 6.25 ± 0.41 µM, pH 6.5,  40°C, [GPP] 0.5 - 25.0 µM used for kinetic parameter determination, kcat 0.09 /s || Gunnewich2008
−
| s<sup>-1</sup>
 
−
| forward
 
−
| ''E. coli'' (E57)
 
−
|
 
−
| <ref name= "Ogura1997"></ref> <ref name="Williams1998"></ref>
 
 
|-
 
|-
−
| 0.0037
+
| Kcat || Forward || GPP || 0.13 || 1/s || in vitro  || 2 || Unrelated  || 0 || E.coli || 4 || Identical (+4) || 4 || close range (+2) || 2 || 36°C - 40°C (+2) || 2 || 128 || Cannabis sativa L., expressed in E.coli,Km of 6.25 ± 0.41 µM, pH 6.5,  40°C, [GPP] 0.5 - 25.0 µM used for kinetic parameter determination, kcat 0.09 /s || Gunnewich2008
−
| s<sup>-1</sup>
 
−
| forward
 
−
| ''E. coli'' (R58)
 
−
|
 
−
| <ref name= "Ogura1997"></ref> <ref name="Williams1998"></ref>
 
 
|-
 
|-
−
| <0.0004
+
| Kcat || Forward || GPP || 0.13 || 1/s || in vitro  || 2 || Unrelated || 0 || E.coli || 4 || Identical (+4) || 4 || close range (+2) || 2 || different temperature (+0) || 0 || 128 || Citrus sinensis, expressed in E.coli 200µM GPP, 20°C, [E]: 0.07 mM, pH7.5 || Entova2013
−
| s<sup>-1</sup>
 
−
| forward
 
−
| ''E. coli'' (R59)
 
−
|
 
−
| <ref name= "Ogura1997"></ref> <ref name="Williams1998"></ref>
 
 
|-
 
|-
−
| <0.0004
+
| Kcat || Forward || GPP || 0.3 || 1/s || in vitro  || 2 || Unrelated  || 0 || E.coli || 4 || Identical (+4) || 4 || 7.0 (+4) || 4 || different temperature (+0) || 0 || 128 || Mentha piperita &amp; Mentha spicata, expressed in E.coli , pH7.0, 30°C, enzyme assay, 1 -10 mg protein per mixture || Alonso1992
−
| s<sup>-1</sup>
 
−
| forward
 
−
| ''E. coli'' (R58P59)
 
−
|
 
−
| <ref name= "Ogura1997"></ref> <ref name="Williams1998"></ref>
 
−
|-
 
−
| <0.0004
 
−
| s<sup>-1</sup>
 
−
| forward
 
−
| ''E. coli'' (R58A59)
 
−
|
 
−
| <ref name= "Ogura1997"></ref> <ref name="Williams1998"></ref>
 
−
|-
 
−
| <0.0004
 
−
| s<sup>-1</sup>
 
−
| forward
 
−
| ''E. coli'' (S60)
 
−
|
 
−
| <ref name= "Ogura1997"></ref> <ref name="Williams1998"></ref>
 
 
|-
 
|-
 +
| Kcat || Forward || GPP || 0.000039 || 1/s || in vitro  || 2 || Unrelated  || 0 || E.coli  || 4 || Identical (+4) || 4 || 7.0 (+4) || 4 || 37°C (+4) || 4 || 512 || Citrus sinensis, expressed in E.coli40mM GPP, 37°C, [E]: 0.3 mM, pH7.0 (check units) || Olsen2011
 
|}
 
|}
  

Revision as of 16:49, 26 April 2016

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 70.03 kDa [1], 72.4 kDa [2] ; 60kDa [3]; 56 kDa [4]

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 [5].

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
10 µM GPP forward Citrus sinensis enzyme activity assay, pH 7, 37°C, with 0.3µM enzyme [6]
40 µM GPP forward Citrus sinensis enzyme activity assay, pH 7, 37°C, with 0.3µM enzyme [6]
0.3 µM Limonene synthase - Citrus sinensis enzyme activity assay, pH 7, 37°C, with 10-40µM GPP [6]
25 µM GPP forward E. coli [7]
10 µM GPP forward Cannabis sativa L. [8]
10 µM GPP forward Mentha sp. [4]
2 µM GPP forward Citrus limon [9]
150 µg Limonene synthase - E. coli [7]
0.07 µM Limonene synthase - Citrus sinensis with 200µM GPP as substrate, at 20°C, pH7.5 [10]
0.26 µM Limonene synthase - Citrus sinensis with 200µM GPP, at 37°C, pH7.5 [10]
200 µM GPP forward Citrus sinensis used for Limonene synthase activity assay at 20°C and 37°C, pH 7.5 [10]
100 µM GPP - Citrus limon [9]
0.1 µM GPP - Citrus limon [9]

Km Values

Parameter Direction Substrate Value Unit Method M_w Organism O_w Expression_vector E_w Enzyme En_w pH P_w Temperature T_w Total weight Description Reference
Km Forward GPP 0.0067 µM in vitro (+2) 2 Unrelated (+0) 0 E.coli (+4) 4 Identical (+4) 4 7.0 (+4) 4 different temperature (+0) 0 128 Native form, protein from Mentha sp. expressed in E. coli. [7]
Km Forward GPP 0.7 µM in vitro (+2) 2 Unrelated (+0) 0 E.coli (+4) 4 Identical (+4) 4 7.0 (+4) 4 different temperature (+0) 0 128 Protein from Citrus limon. Range of 1-100 mM [GPP] was used to determine Km of LimSynth, pH7.0 , 30°C, expressed in E.coli [9]
Km Forward GPP 1.25 µM in vitro (+2) 2 Unrelated (+0) 0 E.coli (+4) 4 Identical (+4) 4 7.0 (+4) 4 37°C (+4) 4 512 5mM [GPP], 32°C, pH7, 10-20 mg protein used for enzyme assay, Ricciocarpos natans [11]
Km Forward GPP 1.8 µM in vitro (+2) 2 Unrelated (+0) 0 E.coli (+4) 4 Identical (+4) 4 7.0 (+4) 4 different temperature (+0) 0 128 From Mentha spicata and Mentha x piperita, pH7.0, 30°C, enzyme assay [4]
Km Forward GPP 6 µM in vitro (+2) 2 Unrelated (+0) 0 E.coli (+4) 4 Identical (+4) 4 7.0 (+4) 4 37°C (+4) 4 512 Protein from Mentha sp., expressed in E.coli, pH7.0, 1-50 mg [E], 25 mM [GPP], limonene produced 94%, 2% myrcene, pinene [7]
Km Forward GPP 6.25 µM in vitro (+2) 2 Unrelated (+0) 0 Phylogenetically related (+2) 2 Identical (+4) 4 close range (+2) 2 36°C - 40°C (+2) 2 64 Protein from Cannabis sativa, expressed in bacteria? Km of 6.25 ± 0.41 mM, pH 6.5, 40°C, [GPP] 0.5 - 25.0 mM used for kinetic parameter determination, kcat 0.09 /s [1]
Km Forward GPP 8.6 µM in vitro (+2) 2 Unrelated (+0) 0 E.coli (+4) 4 Identical (+4) 4 7.0 (+4) 4 37°C (+4) 4 512 protein from Mentha sp., expressed in E.coli, pH7.0, 1-50 mg [E], 25 mM [GPP], limonene produced 94%, 2% myrcene, pinene [7]
Km Forward GPP 11.76 µM in vitro (+2) 2 Unrelated (+0) 0 Phylogenetically related (+2) 2 Identical (+4) 4 7.0 (+4) 4 37°C (+4) 4 256 protein from Cannabis sativa, expressed in bacterial host. Km of 11.76 ± 2.45, pH 7.0, [GPP] 0.5 - 25.0 microM used for kinetic parameter determination. [1]
Km Forward GPP 12.6 µM in vitro (+2) 2 Unrelated (+0) 0 E.coli (+4) 4 Identical (+4) 4 7.0 (+4) 4 37°C (+4) 4 512 Protein from Mentha sp., expressed in E.coli. pH7.0, 1-50 mg [E], 25 mM [GPP], limonene produced 94%, 2% myrcene, pinene [7]
Km Forward GPP 14.9 µM in vitro (+2) 2 Unrelated (+0) 0 E.coli (+4) 4 Identical (+4) 4 7.0 (+4) 4 37°C (+4) 4 512 recombinant preprotein,Protein from Mentha sp., expressed in E.coli. pH7.0, 1-50 mg [E], 25 mM [GPP], limonene produced 94%, 2% myrcene, pinene [7]
Km Forward GPP 16 µM in vitro (+2) 2 Unrelated (+0) 0 E.coli (+4) 4 Identical (+4) 4 7.0 (+4) 4 37°C (+4) 4 512 recombinant preprotein,Protein from Mentha sp., expressed in E.coli. pH7.0, 1-50 mg [E], 25 mM [GPP], limonene produced 94%, 2% myrcene, pinene [7]
Km Forward GPP 18.8 µM in vitro (+2) 2 Unrelated (+0) 0 E.coli (+4) 4 Identical (+4) 4 7.0 (+4) 4 37°C (+4) 4 512 recombinant preprotein,Protein from Mentha sp., expressed in E.coli. pH7.0, 1-50 mg [E], 25 mM [GPP], limonene produced 94%, 2% myrcene, pinene [7]
Km Forward GPP 27.5 µM in vitro (+2) 2 Unrelated (+0) 0 E.coli (+4) 4 Identical (+4) 4 7.0 (+4) 4 37°C (+4) 4 512 recombinant preprotein,Protein from Mentha sp., expressed in E.coli. pH7.0, 1-50 mg [E], 25 mM [GPP], limonene produced 94%, 2% myrcene, pinene [7]
Km Forward GPP 29.9 µM in vitro (+2) 2 Unrelated (+0) 0 E.coli (+4) 4 Identical (+4) 4 7.0 (+4) 4 37°C (+4) 4 512 recombinant preprotein,Protein from Mentha sp., expressed in E.coli. pH7.0, 1-50 mg [E], 25 mM [GPP], limonene produced 94%, 2% myrcene, pinene [7]
Km Forward GPP 32.4 µM in vitro (+2) 2 Unrelated (+0) 0 E.coli (+4) 4 Identical (+4) 4 7.0 (+4) 4 37°C (+4) 4 512 recombinant preprotein,Protein from Mentha sp., expressed in E.coli. pH7.0, 1-50 mg [E], 25 mM [GPP], limonene produced 94%, 2% myrcene, pinene [7]
Km Forward GPP 130 µM in vitro (+2) 2 Unrelated (+0) 0 Phylogenetically related (+2) 2 Identical (+4) 4 7.0 (+4) 4 different temperature (+0) 0 64 from Citrus sinensis, pH 7.5, 20°C, kinetic characterisation assay, [10]

Vmax values

Vmax Unit Directionality Organism Organism References
0.08 µmol/min/mg forward Cannabis sativa L. [1]
0.12 ± 0.01 µmol/min/mg forward Cannabis sativa [1]
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 [10]
0.64 µM/min forward Citrus sinensis 200µM GPP, 37°C, [E]: 0.26µM, pH7.5 [10]
19 µmol/h/mg forward Mentha x piperita & Mentha spicata Maximum specific activity, 1-10µg protein per mixture, pH &.0, 30°C References
Parameter Direction Substrate Value Unit Method M_w Organism O_w Expression_vector E_w Enzyme En_w pH P_w Temperature T_w Total_weight Description Reference
Kcat Forward GPP 0.0004 1/s in vitro (+2) 2 Unrelated (+0) 0 E.coli (+4) 4 Identical (+4) 4 7.0 (+4) 4 different temperature (+0) 0 128 Protein from Mentha sp, expressed in E.coli and truncated (R59), pH7.0, 1-50 mg [E], 25 µM [GPP], limonene produced 94%, 2% myrcene, pinene Williams1998
Kcat Forward GPP 0.0004 1/s in vitro (+2) 2 Unrelated (+0) 0 E.coli (+4) 4 Identical (+4) 4 7.0 (+4) 4 different temperature (+0) 0 128 Protein from Mentha sp. expressed in E.coli , truncated R58P59, pH7.0, 1-50 mg [E], 25 µM [GPP], limonene produced 94%, 2% myrcene, pinene Williams1998
Kcat Forward GPP 0.0004 1/s in vitro (+2) 2 Unrelated (+0) 0 E.coli (+4) 4 Identical (+4) 4 7.0 (+4) 4 different temperature (+0) 0 128 Protein from Mentha sp. expressed in E.coli , truncated R58A59, pH7.0, 1-50 mg [E], 25 µM [GPP], limonene produced 94%, 2% myrcene, pinene Williams1998
Kcat Forward GPP 0.0004 1/s in vitro (+2) 2 Unrelated (+0) 0 E.coli (+4) 4 Identical (+4) 4 7.0 (+4) 4 different temperature (+0) 0 128 Protein from Mentha sp. expressed in E.coli , S60, pH7.0, 1-50 mg [E], 25 µM [GPP], limonene produced 94%, 2% myrcene, pinene Williams1998
Kcat Forward GPP 0.002 1/s in vitro (+2) 2 Unrelated (+0) 0 E.coli (+4) 4 Identical (+4) 4 7.0 (+4) 4 different temperature (+0) 0 128 Protein from Mentha sp. expressed in E.coli preprotein, pH7.0, 1-50 mg [E], 25 µM [GPP], limonene produced 94%, 2% myrcene, pinene Williams1998
Kcat Forward GPP 0.0024 1/s in vitro (+2) 2 Unrelated (+0) 0 E.coli (+4) 4 Identical (+4) 4 7.0 (+4) 4 different temperature (+0) 0 128 Protein from Mentha sp. expressed in E.coli wt, pH7.0, 1-50 mg [E], 25 µM [GPP], limonene produced 94%, 2% myrcene, pinene Williams1998
Kcat Forward GPP 0.0034 1/s in vitro (+2) 2 Unrelated (+0) 0 E.coli (+4) 4 Identical (+4) 4 7.0 (+4) 4 different temperature (+0) 0 128 Protein from Mentha sp. expressed in E.coli E57, pH7.0, 1-50 mg [E], 25 µM [GPP], limonene produced 94%, 2% myrcene, pinene Williams1998
Kcat Forward GPP 0.0036 1/s in vitro 2 Unrelated (+0) 0 E.coli (+4) 4 Identical (+4) 4 7.0 (+4) 4 different temperature (+0) 0 128 Protein from Mentha sp. expressed in E.coli Q54, pH7.0, 1-50 mg [E], 25 µM [GPP], limonene produced 94%, 2% myrcene, pinene Williams1998
Kcat Forward GPP 0.0037 1/s in vitro 2 Unrelated (+0) 0 E.coli 4 Identical (+4) 4 7.0 (+4) 4 different temperature (+0) 0 128 Protein from Mentha sp. expressed in E.coli R58, pH7.0, 1-50 mg [E], 25 µM [GPP], limonene produced 94%, 2% myrcene, pinene Williams1998
Kcat Forward GPP 0.02 1/s in vitro 2 Unrelated 0 E.coli 4 Identical (+4) 4 7.0 (+4) 4 different temperature (+0) 0 128 Protein from Mentha sp. expressed in E.coli recombinant preprotein , pH7.0, 1-50 mg [E], 25 µM [GPP], limonene produced 94%, 2% myrcene, pinene Williams1998
Kcat Forward GPP 0.04 1/s in vitro 2 Unrelated 0 E.coli 4 Identical (+4) 4 close range (+2) 2 37°C (+4) 4 256 Citrus sinensis, expressed in E.coli 200µM GPP, 37°C, [E]: 0.26µM, pH7.5 Entova2013
Kcat Forward GPP 0.082 1/s in vitro 2 Unrelated 0 E.coli 4 Identical (+4) 4 close range (+2) 2 36°C - 40°C (+2) 2 128 Cannabis sativa L., expressed in E.coli,CsTPS1, pH6.5, 40°C, 1.25 mg [LimSynth] per 500ml assay mixture, 10mM [GPP] Gunnewich2008
Kcat Forward GPP 0.09 1/s in vitro 2 Unrelated 0 E.coli 4 Identical (+4) 4 close range (+2) 2 36°C - 40°C (+2) 2 128 Cannabis sativa L., expressed in E.coli,Km of 6.25 ± 0.41 µM, pH 6.5, 40°C, [GPP] 0.5 - 25.0 µM used for kinetic parameter determination, kcat 0.09 /s Gunnewich2008
Kcat Forward GPP 0.13 1/s in vitro 2 Unrelated 0 E.coli 4 Identical (+4) 4 close range (+2) 2 36°C - 40°C (+2) 2 128 Cannabis sativa L., expressed in E.coli,Km of 6.25 ± 0.41 µM, pH 6.5, 40°C, [GPP] 0.5 - 25.0 µM used for kinetic parameter determination, kcat 0.09 /s Gunnewich2008
Kcat Forward GPP 0.13 1/s in vitro 2 Unrelated 0 E.coli 4 Identical (+4) 4 close range (+2) 2 different temperature (+0) 0 128 Citrus sinensis, expressed in E.coli 200µM GPP, 20°C, [E]: 0.07 mM, pH7.5 Entova2013
Kcat Forward GPP 0.3 1/s in vitro 2 Unrelated 0 E.coli 4 Identical (+4) 4 7.0 (+4) 4 different temperature (+0) 0 128 Mentha piperita & Mentha spicata, expressed in E.coli , pH7.0, 30°C, enzyme assay, 1 -10 mg protein per mixture Alonso1992
Kcat Forward GPP 0.000039 1/s in vitro 2 Unrelated 0 E.coli 4 Identical (+4) 4 7.0 (+4) 4 37°C (+4) 4 512 Citrus sinensis, expressed in E.coli40mM GPP, 37°C, [E]: 0.3 mM, pH7.0 (check units) Olsen2011

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. ↑ 1.0 1.1 1.2 1.3 1.4 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
  2. ↑ 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
  3. ↑ 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
  4. ↑ 4.0 4.1 4.2 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
  5. ↑ Latendresse M. (2013). "Computing Gibbs Free Energy of Compounds and Reactions in MetaCyc."
  6. ↑ 6.0 6.1 6.2 Olsen, S.N. 2011. "Isolation, Purification, and Characterization of (+)-4R-limonene synthase: The first step in exploring enzyme stereospecificity in terpenoid biosynthesis",Masters thesis, Brandeis university Cite error: Invalid <ref> tag; name "Olsen2011" defined multiple times with different content Cite error: Invalid <ref> tag; name "Olsen2011" defined multiple times with different content
  7. ↑ 7.00 7.01 7.02 7.03 7.04 7.05 7.06 7.07 7.08 7.09 7.10 7.11 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
  8. ↑ 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
  9. ↑ 9.0 9.1 9.2 9.3 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
  10. ↑ 10.0 10.1 10.2 10.3 10.4 10.5 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
  11. ↑ 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.
  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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