Menthone:Menthol reductase (MMR)

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What we know

Menthone: menthol reductase(s) (MMR) catalyses the NADPH-dependent convertion of menthone to menthol and the conversion of isomenthone to isomenthol.

Issues

Strategies

Reaction catalysed

menthone + NADPH \rightleftharpoons menthol + NADP^+

isomenthone + NADPH \rightleftharpoons isomenthol + NADP^+

Enzyme and 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	BRENDA
menthone:menthol reductase	MMR		34070 Da ^[1], 35000 Da ^[2]		1.1.1.207
menthone
isomenthone
NADPH		C₂₁H₃₀N₇O₁₇P₃	745.42116	16474
NADP⁺		C₂₁H₂₉N₇O₁₇P₃	744.41322	18009

Equation Rate

Two MMR reactions are included in the kinetic model with one converting menthone to menthol and isomenthone, and one converting isomenthone to isomenthol. Both reactions are parameterised using random Bi-Bi reversible Michaelis-Menten equation.

Reaction 1: Conversion of menthone to menthol

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

Reaction 2: Conversion of isomenthone to isomenthol

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

Parameter	Description	Units
V_MMR	Reaction rate for MMR	μM/min
K_{cat_forward}	Catalytic constant in the forward direction	s^-1
Km_menthone	Michaelis-Menten constant for menthone	μM
Km_menthol	Michaelis-Menten constant for menthol	μM
Km_isomenthone	Michaelis-Menten constant for isomenthone	μM
Km_isomenthol	Michaelis-Menten constant for isomenthol	μM
Km_NADPH	Michaelis-Menten constant for NADPH	μM
Km_NADP	Michaelis-Menten constant for NADP⁺	μM
K_eq	Equilibrium constant
[MMR]	enzyme concentration	μM
[menthol]	menthol concentration	μM
[menthone]	Menthone concentration	μM
[isomenthone]	Isomenthone concentration	μM
[isomenthol]	Isomenthol concentration	μM
[NADPH]	NADPH concentration	μM
[NADP]	NADP⁺ concentration	μM

Strategies for estimating the kinetic parameter values

Estimating parameters for MMR

Standard Gibbs Free energy

The Gibbs free energy for MMR is -1.6264343 kcal.mol^-1. This value is estimated from the 'Contribution group' method by Latendresse, M. and is available from MetaCyc (EC 1.1.1.207) ^[3].

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	menthone	69.9	µM	From Helen's data, would need to ask for experimental conditions, unpublished. Assume optimal conditions. NEEDS CONFIRMING	Toogood2015^[4]
Km	Forward	menthone	73.8	µM	KM: 69.9 +/- 3.9. From Helen's data, would need to ask for experimental conditions, unpublished. Assume optimal conditions. NEEDS CONFIRMING	Toogood2015^[4]
Km	Forward	menthone	66	µM	KM: 69.9 +/- 3.9. From Helen's data, would need to ask for experimental conditions, unpublished. Assume optimal conditions. NEEDS CONFIRMING	Toogood2015^[4]
Km	Forward	isomenthone	86.4	µM	Km: 86.4 +/- 5.4. From Helen's data, would need to ask for experimental conditions, unpublished. Assume optimal conditions. NEEDS CONFIRMING	Toogood2015^[4]
Km	Forward	isomenthone	91.8	µM	Km: 86.4 +/- 5.4. From Helen's data, would need to ask for experimental conditions, unpublished. Assume optimal conditions. NEEDS CONFIRMING	Toogood2015^[4]
Km	Forward	isomenthone	81	µM	Km: 86.4 +/- 5.4. From Helen's data, would need to ask for experimental conditions, unpublished. Assume optimal conditions. NEEDS CONFIRMING	Toogood2015^[4]
Km	Forward	menthone	3	µM	menthone + nadph -> menthol, Km: 3.0 +/- 0.6, pH 7.0, temp 31°C, from Mentha piperita, expressed in E.coli.	Davis2005 ^[1]
Km	Forward	menthone	3.6	µM	menthone + nadph -> menthol, Km: 3.0 +/- 0.6, pH 7.0, temp 31°C, from Mentha piperita, expressed in E.coli.	Davis2005 ^[1]
Km	Forward	menthone	2.4	µM	menthone + nadph -> menthol, Km: 3.0 +/- 0.6, pH 7.0, temp 31°C, from Mentha piperita, expressed in E.coli.	Davis2005 ^[1]
Km	Forward	isomenthone	41	µM	isomenthone + nadph -> menthol, Km: 41.0 +/- 5, pH 7.0, temp 31°C, from Mentha piperita, expressed in E.coli.	Davis2005 ^[1]
Km	Forward	isomenthone	46	µM	isomenthone + nadph -> menthol, Km: 41.0 +/- 5.0, pH 7.0, temp 31°C, from Mentha piperita, expressed in E.coli.	Davis2005 ^[1]
Km	Forward	isomenthone	35	µM	isomenthone + nadph -> menthol, Km: 41.0+/- 5.0, pH 7.0, temp 31°C, from Mentha piperita, expressed in E.coli.	Davis2005 ^[1]
Km	Forward	NADPH	0.12	µM	isomenthone + nadph -> menthol, Km: 0.12+/-0.04, pH 7.0 temp 31°C, from Mentha piperita, expressed in E.coli.	Davis2005 ^[1]
Km	Forward	NADPH	0.16	µM	isomenthone + nadph -> menthol, Km: 0.12+/- 0.04 pH 7.0 temp 31°C, from Mentha piperita, expressed in E.coli.	Davis2005 ^[1]
Km	Forward	NADPH	0.08	µM	isomenthone + nadph -> menthol, Km: 0.12+/- 0.04, pH 9.3, temp 31°C, from Mentha piperita, expressed in E.coli.	Davis2005 ^[1]
Km	Forward	menthone	250	µM	menthone -> menthol, pH 7.5, 30C, from Mentha piperita, expressed in E. coli	Kjonaas1982 ^[2]
Km	Forward	NADPH	20	µM	menthone -> menthol, pH 7.5, 30C, from Mentha piperita, expressed in E. coli	Kjonaas1982 ^[2]

Kcat values

Parameter	Directionality	Substrate / Product	Value	unit	Method notes	References
Kcat	Forward	menthone	2.34	s^-1	Kcat: 2.34 +/- 0.05. From Helen's data, would need to ask for experimental conditions, unpublished. Assume optimal conditions. NEEDS CONFIRMING	Toogood2015^[4]
Kcat	Forward	menthone	2.29	s^-1	Kcat: 2.34 +/- 0.05. From Helen's data, would need to ask for experimental conditions, unpublished. Assume optimal conditions. NEEDS CONFIRMING	Toogood2015^[4]
Kcat	Forward	menthone	2.39	s^-1	Kcat: 2.34 +/- 0.05. From Helen's data, would need to ask for experimental conditions, unpublished. Assume optimal conditions. NEEDS CONFIRMING	Toogood2015^[4]
Kcat	Forward	isomenthone	2.28	s^-1	Kcat: 2.28 +/- 0.06. From Helen's data, would need to ask for experimental conditions, unpublished. Assume optimal conditions. NEEDS CONFIRMING	Toogood2015^[4]
Kcat	Forward	isomenthone	2.22	s^-1	Kcat: 2.28 +/- 0.06. From Helen's data, would need to ask for experimental conditions, unpublished. Assume optimal conditions. NEEDS CONFIRMING	Toogood2015^[4]
Kcat	Forward	isomenthone	2.34	s^-1	Kcat: 2.28 +/- 0.06. From Helen's data, would need to ask for experimental conditions, unpublished. Assume optimal conditions. NEEDS CONFIRMING	Toogood2015^[4]
Kcat	Forward	menthone/isomenthone	0.6	s^-1	isomenthone + nadph -> menthol, Kcat: 0.6, pH 7.0, temp 31°C, from Mentha piperita, expressed in E.coli.	Davis2005 ^[1]

Parameter uncertainty

Probability distribution has been generated for each of the parameter in this reaction.

**Values and weights of Km for isomenthone**
Parameter	Direction	Substrate	Value	Unit	Weight	weight counter	Probability distribution	Parameter details
Km	Forward	isomenthone	35	µM	512	512		Mode: 63.5
Km	Forward	isomenthone	41	µM	512	1024		Value Range: 41 - 91.8 μM
Km	Forward	isomenthone	46	µM	512	1536		CI factor: 1.497
Km	Forward	isomenthone	81	µM	512	2048		μ:4.1902
Km	Forward	isomenthone	86.4	µM	512	2560		σ: 0.19798
Km	Forward	isomenthone	91.8	µM	512	3072

**Values and weights of Km for menthone**
Parameter	Direction	Substrate	Value	Unit	Weight	weight counter	Probability distribution	Parameter details
Km	Forward	menthone	2.4	µM	512	512		Mode: 34.8
Km	Forward	menthone	3	µM	512	1024		Value Range: 3.7 - 326.84 μM
Km	Forward	menthone	3.6	µM	512	1536		CI factor: 9.39
Km	Forward	menthone	66	µM	512	2048		μ:4.31152
Km	Forward	menthone	69.9	µM	512	2560		σ:0.87287
Km	Forward	menthone	73.8	µM	512	3072
Km	Forward	menthone	250	µM	256	3328

**Values and weights of Km for NADPH**
Parameter	Direction	Substrate	Value	Unit	Weight	weight counter	Probability distribution	Parameter details
Km	Forward	NADPH	0.12	µM	512	512		Mode: 0.14
Km	Forward	NADPH	0.16	µM	512	1024		Value Range: 0.0019 - 10.1225 μM
Km	Forward	NADPH	0.08	µM	512	1536		CI factor: 72.304
Km	Forward	NADPH	20	µM	256	1792		μ:-0.0343, σ:1.3898

**Values and weights of Kcat for Menthone**
Parameter	Direction	Substrate	Value	Unit	Weight	weight counter	Probability distribution	Parameter details
Kcat	Forward	menthone	0.6	s^-1	512	512		Mode: 2.35
Kcat	Forward	menthone	2.29	s^-1	512	1024		Value Range: 0.9468 - 5.6602 μM
Kcat	Forward	menthone	2.34	s^-1	512	1536		CI factor: 2.445
Kcat	Forward	menthone	2.39	s^-1	512	2048		μ:1.0108, σ:0.414

**Values and weights of Kcat for isomenthone**
Parameter	Direction	Substrate	Value	Unit	Weight	weight counter	Probability distribution	Parameter details
Kcat	Forward	isomenthone	0.6	1/s	512	512		Mode: 2.25
Kcat	Forward	isomenthone	2.22	1/s	512	1024		Value Range: 0.9395 - 5.3887 μM
Kcat	Forward	isomenthone	2.28	1/s	512	1536		CI factor: 2.395
Kcat	Forward	isomenthone	2.34	1/s	512	2048		μ:0.9754, σ:0.4055

Detailed description of kinetic values obtained from literature

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

Simulations

References

↑ ^1.00 ^1.01 ^1.02 ^1.03 ^1.04 ^1.05 ^1.06 ^1.07 ^1.08 ^1.09 ^1.10 Davis, E.M. et. al. (2005). "Monoterpene metabolism. Cloning, Expression, and Characterization of Menthone Reductases from Peppermint.", Vol 137, pp. 873-881
↑ ^2.0 ^2.1 ^2.2 Kjonaas, R. et. al. (1982). Metabolism of monoterpenes: Conversion of l-Menthone to l-Menthol and d-Neomenthol by stereospecific dehydrogenases from peppermint (Mentha piperita) leaves, vol 69, pp.1013-1017.
↑ Latendresse M. (2013). "Computing Gibbs Free Energy of Compounds and Reactions in MetaCyc."
↑ ^4.00 ^4.01 ^4.02 ^4.03 ^4.04 ^4.05 ^4.06 ^4.07 ^4.08 ^4.09 ^4.10 ^4.11 Toogood, H.S. et. al. (2015). "Enzymatic Menthol Production: One-Pot Approach Using Engineered Escherichia coli", ACS Synth Biol Vol 4, pp. 1112-1123

[Davis2005-1] 1.00 ^1.01 ^1.02 ^1.03 ^1.04 ^1.05 ^1.06 ^1.07 ^1.08 ^1.09 ^1.10 Davis, E.M. et. al. (2005). "Monoterpene metabolism. Cloning, Expression, and Characterization of Menthone Reductases from Peppermint.", Vol 137, pp. 873-881

[Kjonaas1982-2] 2.0 ^2.1 ^2.2 Kjonaas, R. et. al. (1982). Metabolism of monoterpenes: Conversion of l-Menthone to l-Menthol and d-Neomenthol by stereospecific dehydrogenases from peppermint (Mentha piperita) leaves, vol 69, pp.1013-1017.

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

[Toogood2015-4] 4.00 ^4.01 ^4.02 ^4.03 ^4.04 ^4.05 ^4.06 ^4.07 ^4.08 ^4.09 ^4.10 ^4.11 Toogood, H.S. et. al. (2015). "Enzymatic Menthol Production: One-Pot Approach Using Engineered Escherichia coli", ACS Synth Biol Vol 4, pp. 1112-1123

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Menthone:Menthol reductase (MMR)

Contents

What we know

Issues

Strategies

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

Kcat values

Parameter uncertainty

Detailed description of kinetic values obtained from literature

Simulations

References

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