Difference between revisions of "Double-bond reductase (DBR)"

Latest revision as of 13:36, 23 March 2017

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

Reaction catalysed

Enzyme and Metabolite Background Information

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

Compound	Abbreviation	Chemical Formula	Molar mass (g/mol)	ChEBI	BRENDA
Double-bond reductase	DBR		37914 Da		1.3.1.81
pulegone		C₁₀H₁₆O	136.24
menthone
NADPH		C₂₁H₃₀N₇O₁₇P₃	745.42116	16474
NADP⁺		C₂₁H₂₉N₇O₁₇P₃	744.41322	18009
isomenthone

Equation Rate

DBR is modelled using two equations that describes: 1) Pulegone to Menthone and 2) Pulegone to Isomenthone. The predominant product for DBR is menthone where the ratio of menthone to isomenthone production have been reported as 40:33 ^[1], 55:45 ^[2] and 70:30 ^[3]. To model the variability of the product yield for DBR, variability factors which are calculated from the average ratio is included in the equations.

^[1]

Reaction 1: Conversion of pulegone to menthone

V_\mathrm{DBR} = Kcat_\mathrm{forward} * X1*[DBR] * \cfrac {\left ( \cfrac{[pulegone]}{Km_\mathrm{pulegone}} * \cfrac {[NADPH]}{Km_\mathrm{NADPH}} \right ) * \left ( 1 - \cfrac {[menthone]*[NADP]}{[pulegone]*[NADPH]*K_\mathrm{eq}} \right )} { \left (1 + \cfrac {[Pulegone]}{Km_\mathrm{Pulegone}} + \cfrac {[NADP]}{Km_\mathrm{NADP}} \right ) + \left ( 1+ \cfrac {[NADPH]}{Km_\mathrm{NADPH}} + \cfrac {[menthone]}{Km_\mathrm{menthone}} \right ) }

Reaction 2: Conversion of pulegone to isomenthone

V_\mathrm{DBR} = Kcat_\mathrm{forward} *X2* [DBR] * \cfrac {\left ( \cfrac{[pulegone]}{Km_\mathrm{pulegone}} * \cfrac {[NADPH]}{Km_\mathrm{NADPH}} \right ) * \left ( 1 - \cfrac {[isomenthone]*[NADP]}{[pulegone]*[NADPH]*K_\mathrm{eq}} \right )} { \left (1 + \cfrac {[Pulegone]}{Km_\mathrm{Pulegone}} + \cfrac {[NADP]}{Km_\mathrm{NADP}} \right ) + \left ( 1+ \cfrac {[NADPH]}{Km_\mathrm{NADPH}} + \cfrac {[isomenthone]}{Km_\mathrm{isomenthone}} \right ) }

Parameter	Description	Units
V_PGR	Reaction rate for Limonene-3-hydroxylase	μM/min
K_{cat_forward}	Catalytic constant in the forward direction	s^-1
Km_pulegone	Michaelis-Menten constant for pulegone	μM
Km_menthone	Michaelis-Menten constant for menthone	μM
Km_isomenthone	Michaelis-Menten constant for isomenthone	μM
Km_NADPH	Michaelis-Menten constant for NADPH	μM
Km_NADP	Michaelis-Menten constant for NADP⁺	μM
K_eq	Equilibrium constant
X1 , X2	Variability factors
[PGR]	enzyme concentration	μM
[pulegone]	Pulegone concentration	μM
[menthone]	Menthone concentration	μM
[isomenthone]	Isomenthone concentration	μM
[NADPH]	NADPH concentration	μM
[NADP]	NADP⁺ concentration	μM

Strategies for estimating the kinetic parameter values

Standard Gibbs Free energy

The Gibbs free energy for PGR is -3.9565125 kcal.mol^-1. This value is estimated from the 'Contribution group' method by Latendresse, M. and is available from MetaCyc (EC 1.3.1.81) ^[4].

Calculating 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 )

$= exp \left ( \cfrac {-(XY \text { kJmol}^{-1})}{ (8.31 \text{ JK}^{-1} \text { mol}^{-1} * 289 K} \right )$

$= exp \left ( \cfrac { XY \text { kJmol}^{-1} }{ 2401.59 \text{ JK}^{-1}\text { mol}^{-1} }\right)$

$= exp \left ( \cfrac{ XY \text { Jmol}^{-1}}{2401.59 \text{ JK}^{-1}\text { mol}^{-1}} \right)$

$=exp \left ( XY \right )$

$= (INSERT RESULT)$

where;

K_eq	Equilibrium constant
-ΔG^°	Gibbs free energy change. For (INSERT ENZYME) it is (INSERT VALUE) kJmol^-1
R	Gas constant with a value of 8.31 JK^-1mol^-1
T	Temperature which is always expressed in kelvin

Extracting Information from menthone Production Rates

A table will go here

Published Kinetic Parameter Values

Km Values

Parameter	Directionality	Substrate / Product	Value	unit	Method notes	References
Km	Forward	pulegone	2.3	µM	Gene from peppermint oil gland secretory cell cDNA, expressed in E. coli, optimal pH 5.0, menthone:isomenthone ratio of 55:45	Ringer2003
Km	Forward	pulegone	2.9	µM	Gene from peppermint oil gland secretory cell cDNA, expressed in E. coli, optimal pH 5.0, menthone:isomenthone ratio of 55:45, Km 2.3 +/- 0.6	Ringer2003
Km	Forward	NADPH	6.9	µM	Gene from peppermint oil gland secretory cell cDNA, expressed in E. coli, optimal pH 5.0, menthone:isomenthone ratio of 55:45	Ringer2003

Detailed description of kinetic values obtained from literature

A more detailed description of the values listed above can be found here .

Simulations

The DBR model have been used to simulate the biotransformation experiment from published experiments. Click on the pictures below to learn more:

References

↑ ^1.0 ^1.1 Toogood, H. et. al.2015. "Enzymatic menthol production: One-pot approach using engioneered Escherichia coli ", ACS Synthetic Biology, 4:1112-1123 Cite error: Invalid <ref> tag; name "Toogood2015" defined multiple times with different content
↑ Ringer, K.L. et. al. 2003. "Monoterpene double-bond reductases of the (-)-menthol biosynthetic pathway:isolation and characterization of cDNAs encoding (-)-isopiperitenone reductase and (+)-pulegone reductase of peppermint", Archives of Biochemistry and Biophysics 418:80-92
↑ Solodar, J. 1976. "Stereoselective reduction of menthone and isomenthone by dissolving alkali metals and by hydrogen with Group 8 Metals on carbon", The Journal of Organic Chemistry, 41(21):3461-3464
↑ Latendresse M. (2013). "Computing Gibbs Free Energy of Compounds and Reactions in MetaCyc."

[Toogood2015-1] 1.0 ^1.1 Toogood, H. et. al.2015. "Enzymatic menthol production: One-pot approach using engioneered Escherichia coli ", ACS Synthetic Biology, 4:1112-1123 Cite error: Invalid <ref> tag; name "Toogood2015" defined multiple times with different content

[Ringer2003-2] Ringer, K.L. et. al. 2003. "Monoterpene double-bond reductases of the (-)-menthol biosynthetic pathway:isolation and characterization of cDNAs encoding (-)-isopiperitenone reductase and (+)-pulegone reductase of peppermint", Archives of Biochemistry and Biophysics 418:80-92

[Solodar1976-3] Solodar, J. 1976. "Stereoselective reduction of menthone and isomenthone by dissolving alkali metals and by hydrogen with Group 8 Metals on carbon", The Journal of Organic Chemistry, 41(21):3461-3464

[4] Latendresse M. (2013). "Computing Gibbs Free Energy of Compounds and Reactions in MetaCyc."

[1]

[2]

[3]

[4]

@@ Line 1: / Line 1: @@
 You can go back to main page of the kinetic model [http://www.systemsbiology.ls.manchester.ac.uk/wiki/index.php/Kinetic_Model_of_Monoterpenoid_Biosynthesis_Wiki here].
-'''Legend''':
-{|
-|-
-|Have not started ··
-|1 -2 data found ··
-|3-4 data found ··
-|sufficient data found/estimated ··
-|data distribution generated ··
-|data sampled
-|-
-|style="border-bottom:1px solid #abd5f5; background:#d0e5f5; padding:0.2em 0.5em; font-size:110%; font-weight:bold; text-align:center;"|to do
-|style="background-color: #8CF506;" |
-|style="background-color: #F5F106;" |
-|style="background-color: #FFC300;" |
-|style="background-color: #FF5733;" |
-|style="background-color: #C70039; font-size:110%; font-weight:bold; color:white; text-align:center;" | DONE!
-|}
-== What we know ==
-Pulegone reductase(s) (PGR) catalyses the NADPH-dependent convertion of pulegone to menthone and isomenthone (the former predominates).
-=== Issues ===
-=== Strategies ===
 == Reaction catalysed ==
+[[File:DBR_reaction_diagram_v2.png | left | 500px ]]
-:<math>
-pulegone + NADPH  \rightleftharpoons menthone + NADP^+
-</math>
-:<math>
-pulegone + NADPH  \rightleftharpoons isomenthone + NADP^+
-</math>
 == Enzyme and Metabolite Background Information ==
@@ Line 49: / Line 12: @@
 {| class="wikitable" style="text-align:center"
 |-
-! style="border: 1px solid black; padding: 5px; background: #ADD8E6;"|Metabolite
+! style="border: 1px solid black; padding: 5px; background: #ADD8E6;"|Compound
 ! style="border: 1px solid black; padding: 5px; background: #ADD8E6;"|Abbreviation
 ! style="border: 1px solid black; padding: 5px; background: #ADD8E6;"|Chemical Formula
@@ Line 59: / Line 22: @@
 ! style="border: 1px solid black; padding: 5px; background: #ADD8E6;"|PlantCyc
 |-
-| pulegone reductase
+| Double-bond reductase
-| PGR
+| DBR
 |
 | 37914 Da
@@ Line 123: / Line 86: @@
 == Equation Rate ==
-Two PGR reactions are included in the kinetic model with each converting pulegone to methone and isomenthone respectively. Both reactions are parameterised using random Bi-Bi reversible Michaelis-Menten equation.
+DBR is modelled using two equations that describes: 1) Pulegone to Menthone and 2) Pulegone to Isomenthone. The predominant product for DBR is menthone where the ratio of menthone to isomenthone production have been reported as 40:33 <ref name="Toogood2015"> [http://www.ncbi.nlm.nih.gov/pubmed/26017480 Toogood, H. ''et. al.''2015. "Enzymatic menthol production: One-pot approach using engioneered ''Escherichia coli'' "], ACS Synthetic Biology, '''4''':1112-1123</ref>, 55:45 <ref name="Ringer2003">[http://www.ncbi.nlm.nih.gov/pubmed/13679086 Ringer, K.L. ''et. al.'' 2003. "Monoterpene double-bond reductases of the (-)-menthol biosynthetic pathway:isolation and characterization of cDNAs encoding (-)-isopiperitenone reductase and (+)-pulegone reductase of peppermint"], Archives of Biochemistry and Biophysics '''418''':80-92</ref> and 70:30 <ref name="Solodar1976">[http://pubs.acs.org/doi/abs/10.1021/jo00883a031 Solodar, J. 1976. "Stereoselective reduction of menthone and isomenthone by dissolving alkali metals and by hydrogen with Group 8 Metals on carbon"], The Journal of Organic Chemistry, '''41'''(21):3461-3464</ref>. To model the variability of the product yield for DBR, variability factors which are calculated from the average ratio is included in the equations.
+<ref name="Toogood2015"> [http://www.ncbi.nlm.nih.gov/pubmed/26017480 Toogood, H. ''et. al.'']2015. "Enzymatic menthol production: One-pot approach using engioneered ''Escherichia coli'' ", ACS Synthetic Biology, '''4''':1112-1123</ref>
 ''' Reaction 1: Conversion of pulegone to menthone'''
 :<math>
-V_\mathrm{PGR} =  Kcat_\mathrm{forward} * [PGR] * \cfrac {\left ( \cfrac{[pulegone]}{Km_\mathrm{pulegone}} * \cfrac {[NADPH]}{Km_\mathrm{NADPH}} \right ) * \left ( 1 - \cfrac {[menthone]*[NADP]}{[pulegone]*[NADPH]*K_\mathrm{eq}} \right )}
+V_\mathrm{DBR} =  Kcat_\mathrm{forward} * X1*[DBR] * \cfrac {\left ( \cfrac{[pulegone]}{Km_\mathrm{pulegone}} * \cfrac {[NADPH]}{Km_\mathrm{NADPH}} \right ) * \left ( 1 - \cfrac {[menthone]*[NADP]}{[pulegone]*[NADPH]*K_\mathrm{eq}} \right )}
-{ \left (1 + \cfrac {[NADPH]}{Km_\mathrm{NADPH}} + \cfrac {[NADP]}{Km_\mathrm{NADP}} \right )  + \left ( 1+ \cfrac {[pulegone]}{Km_\mathrm{pulegone}}  + \cfrac {[menthone]}{Km_\mathrm{menthone}} \right ) }
+{ \left (1 + \cfrac {[Pulegone]}{Km_\mathrm{Pulegone}} + \cfrac {[NADP]}{Km_\mathrm{NADP}} \right )  + \left ( 1+ \cfrac {[NADPH]}{Km_\mathrm{NADPH}}  + \cfrac {[menthone]}{Km_\mathrm{menthone}} \right ) }
 </math>
 ''' Reaction 2: Conversion of pulegone to isomenthone'''
 :<math>
-V_\mathrm{PGR} =  Kcat_\mathrm{forward} * [PGR] * \cfrac {\left ( \cfrac{[pulegone]}{Km_\mathrm{pulegone}} * \cfrac {[NADPH]}{Km_\mathrm{NADPH}} \right ) * \left ( 1 - \cfrac {[isomenthone]*[NADP]}{[pulegone]*[NADPH]*K_\mathrm{eq}} \right )}
+V_\mathrm{DBR} =  Kcat_\mathrm{forward} *X2* [DBR] * \cfrac {\left ( \cfrac{[pulegone]}{Km_\mathrm{pulegone}} * \cfrac {[NADPH]}{Km_\mathrm{NADPH}} \right ) * \left ( 1 - \cfrac {[isomenthone]*[NADP]}{[pulegone]*[NADPH]*K_\mathrm{eq}} \right )}
-{ \left (1 + \cfrac {[NADPH]}{Km_\mathrm{NADPH}} + \cfrac {[NADP]}{Km_\mathrm{NADP}} \right )  + \left ( 1+ \cfrac {[pulegone]}{Km_\mathrm{pulegone}}  + \cfrac {[isomenthone]}{Km_\mathrm{isomenthone}} \right ) }
+{ \left (1 + \cfrac {[Pulegone]}{Km_\mathrm{Pulegone}} + \cfrac {[NADP]}{Km_\mathrm{NADP}} \right )  + \left ( 1+ \cfrac {[NADPH]}{Km_\mathrm{NADPH}}  + \cfrac {[isomenthone]}{Km_\mathrm{isomenthone}} \right ) }
 </math>
@@ Line 157: / Line 122: @@
 |-
 | K<sub>eq</sub> || Equilibrium constant ||
+|-
+| X1 , X2 || Variability factors ||
 |-
 | [PGR] || enzyme concentration || μM
@@ Line 230: / Line 197: @@
 |}
-=== Extracting Information from (INSERT SUBSTRATE/PRODUCT) Production Rates ===
+=== Extracting Information from menthone Production Rates ===
 A table will go here
@@ Line 236: / Line 203: @@
 === Published Kinetic Parameter Values ===
-A table will go here.
+==== Km Values ====
+{|
+! style="border: 1px solid black; padding: 5px; background: #ffdead; width: 50px;" | Parameter
+! style="border: 1px solid black; padding: 5px; background: #ffdead; width: 100px;" | Directionality
+! style="border: 1px solid black; padding: 5px; background: #ffdead; width: 100px;" | Substrate / Product
+! style="border: 1px solid black; padding: 5px; background: #ffdead; width: 50px;" | Value
+! style="border: 1px solid black; padding: 5px; background: #ffdead; width: 50px;" | unit
+! style="border: 1px solid black; padding: 5px; background: #ffdead; width: 300px;" | Method notes
+! style="border: 1px solid black; padding: 5px; background: #ffdead; width: 10px;" | References
+|-
+|  Km || Forward || pulegone || 2.3 || µM || Gene from peppermint oil gland secretory cell cDNA, expressed in E. coli, optimal pH 5.0, menthone:isomenthone ratio of 55:45 || Ringer2003
+|-
+| Km || Forward || pulegone || 2.9 || µM || Gene from peppermint oil gland secretory cell cDNA, expressed in E. coli, optimal pH 5.0, menthone:isomenthone ratio of 55:45, Km 2.3 +/- 0.6 || Ringer2003
+|-
+|  Km || Forward || NADPH || 6.9 || µM || Gene from peppermint oil gland secretory cell cDNA, expressed in E. coli, optimal pH 5.0, menthone:isomenthone ratio of 55:45 || Ringer2003
+|}
 === Detailed description of kinetic values obtained from literature ===
@@ Line 245: / Line 226: @@
 == Simulations ==
+The DBR model have been used to simulate the biotransformation experiment from published experiments. Click on the pictures below to learn more:
+<imagemap>
+File:DBR_biotrans_toogood_simulation.png| 500px
+rect 41 49 941 684 [[Simulating DBR Biotransformation]]
+desc bottom-left
+</imagemap>
 == References ==
 <references />

Difference between revisions of "Double-bond reductase (DBR)"

Latest revision as of 13:36, 23 March 2017

Contents

Reaction catalysed

Enzyme and Metabolite Background Information

Equation Rate

Strategies for estimating the kinetic parameter values

Standard Gibbs Free energy

Calculating the Equilibrium Constant

Extracting Information from menthone Production Rates

Published Kinetic Parameter Values

Km Values

Detailed description of kinetic values obtained from literature

Simulations

References

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