Menthone:Neomenthol reductase (MNMR)

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What we know

Menthone: neomenthol reductase(s) (MNMR) catalyses the NADPH-dependent convertion of menthone to neomenthol and the conversion of isomenthone to neoisomenthol.

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 neomenthol, and one converting isomenthone to neoisomenthol. Both reactions are parameterised using random Bi-Bi reversible Michaelis-Menten equation.

Reaction 1: Conversion of menthone to neomenthol

V_\mathrm{MNMR} = Kcat_\mathrm{forward} * [MNMR] * \cfrac {\left ( \cfrac{[menthone]}{Km_\mathrm{menthone}} * \cfrac {[NADPH]}{Km_\mathrm{NADPH}} \right ) * \left ( 1 - \cfrac {[neomenthol]*[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 {[neomenthol]}{Km_\mathrm{neomenthol}} \right ) }

Reaction 2: Conversion of isomenthone to neoisomenthol

V_\mathrm{MNMR} = Kcat_\mathrm{forward} * [MNMR] * \cfrac {\left ( \cfrac{[isomenthone]}{Km_\mathrm{isomenthone}} * \cfrac {[NADPH]}{Km_\mathrm{NADPH}} \right ) * \left ( 1 - \cfrac {[neoisomenthol]*[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 {[neoisomenthol]}{Km_\mathrm{neoisomenthol}} \right ) }

Parameter	Description	Units
V_MMR	Reaction rate for MNMR	μM/min
K_{cat_forward}	Catalytic constant in the forward direction	s^-1
Km_menthone	Michaelis-Menten constant for menthone	μM
Km_neomenthol	Michaelis-Menten constant for neomenthol	μM
Km_isomenthone	Michaelis-Menten constant for isomenthone	μM
Km_{neoisomenthol}	Michaelis-Menten constant for neoisomenthol	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
[neomenthol]	neomenthol concentration	μM
[menthone]	Menthone concentration	μM
[isomenthone]	Isomenthone concentration	μM
[neoisomenthol]	neoisomenthol 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 for menthone, isomenthone, neomenthol, neoisomenthol and NADPH

Parameter	Directionality	Substrate / Product	Value	unit	Method notes	References
Km	Forward	menthone	96	µM	Km: 96.0 +/- 5.2. From Helen's data, would need to ask for experimental conditions, unpublished. Assume optimal conditions. NEEDS CONFIRMING	Toogood2015^[4]
Km	Forward	menthone	101.2	µM	Km: 96.0 +/- 5.2. From Helen's data, would need to ask for experimental conditions, unpublished. Assume optimal conditions. NEEDS CONFIRMING	Toogood2015^[4]
Km	Forward	menthone	90.8	µM	Km: 96.0 +/- 5.2. From Helen's data, would need to ask for experimental conditions, unpublished. Assume optimal conditions. NEEDS CONFIRMING	Toogood2015^[4]
Kcat	Forward	menthone	0.91	1/s	Kcat: 0.89 +/- 0.02. From Helen's data, would need to ask for experimental conditions, unpublished. Assume optimal conditions. NEEDS CONFIRMING	Toogood2015^[4]
Kcat	Forward	menthone	0.87	1/s	Kcat: 0.89 +/- 0.02. From Helen's data, would need to ask for experimental conditions, unpublished. Assume optimal conditions. NEEDS CONFIRMING	Toogood2015^[4]
Kcat	Forward	isomenthone	0.44	1/s	Kcat: 0.44 +/- 0.02. From Helen's data, would need to ask for experimental conditions, unpublished. Assume optimal conditions. NEEDS CONFIRMING	Toogood2015^[4]
Kcat	Forward	isomenthone	0.46	1/s	Kcat: 0.44 +/- 0.02. From Helen's data, would need to ask for experimental conditions, unpublished. Assume optimal conditions. NEEDS CONFIRMING	Toogood2015^[4]
Kcat	Forward	isomenthone	0.42	1/s	Kcat: 0.44 +/- 0.02. From Helen's data, would need to ask for experimental conditions, unpublished. Assume optimal conditions. NEEDS CONFIRMING	Toogood2015^[4]
Km	Forward	isomenthone	186.5	µM	Km: 186.5 +/- 16.8. From Helen's data, would need to ask for experimental conditions, unpublished. Assume optimal conditions. NEEDS CONFIRMING	Toogood2015^[4]
Km	Forward	isomenthone	203.3	µM	Km: 186.5 +/- 16.8. From Helen's data, would need to ask for experimental conditions, unpublished. Assume optimal conditions. NEEDS CONFIRMING	Toogood2015^[4]
Km	Forward	isomenthone	169.7	µM	Km: 186.5 +/- 16.8. From Helen's data, would need to ask for experimental conditions, unpublished. Assume optimal conditions. NEEDS CONFIRMING	Toogood2015^[4]
Km	Forward	menthone	7.1	µM	producing neomenthol. KM: 7.1 +/- 1.4. from Artemisia annua, pH 7.0, Temp 30C,	Ryden2010^[5]
Km	Forward	menthone	8.5	µM	producing neomenthol. KM: 7.1 +/- 1.4. from Artemisia annua, pH 7.0, Temp 30C,	Ryden2010^[5]
Km	Forward	menthone	5.7	µM	producing neomenthol. KM: 7.1 +/- 1.4. from Artemisia annua, pH 7.0, Temp 30C,	Ryden2010^[5]
Km	Reverse	neomenthol	305	µM	producing menthone from neomenthol (reverse). KM: 305 +/- 100. from Artemisia annua, pH 7.0, Temp 30C,	Ryden2010^[5]
Km	Reverse	neomenthol	405	µM	producing menthone from neomenthol (reverse). KM: 305 +/- 100. from Artemisia annua, pH 7.0, Temp 30C,	Ryden2010^[5]
Km	Reverse	neomenthol	205	µM	producing menthone from neomenthol (reverse). KM: 305 +/- 100. from Artemisia annua, pH 7.0, Temp 30C,	Ryden2010^[5]
Kcat	Forward	menthone	0.6	1/s	producing neomenthol (reverse). Kcat: 0.60. from Artemisia annua, pH 7.0, Temp 30C,	Ryden2010^[5]
Kcat	Reverse	neomenthol	0.91	1/s	producing neomenthol (reverse). Kcat: 0.91. from Artemisia annua, pH 7.0, Temp 30C,	Ryden2010^[5]
Km	Forward	menthone	674	µM	menthone -> neomenthone, Km: 674 +/- 78, pH 9.3, temp 30 c, from Mentha piperita, expressed in E.coli.	Davis2005^[1]
Km	Forward	menthone	752	µM	menthone -> neomenthone, Km: 674 +/- 78, pH 9.3, temp 30 c, from Mentha piperita, expressed in E.coli.	Davis2005^[1]
Km	Forward	menthone	596	µM	menthone -> neomenthone, Km: 674 +/- 78, pH 9.3, temp 30 c, from Mentha piperita, expressed in E.coli.	Davis2005^[1]
Km	Forward	isomenthone	1000	µM	isomenthone -> neomenthone, Km: >1000, pH 9.3, temp 30 c, from Mentha piperita, expressed in E.coli.	Davis2005^[1]
Km	Forward	NADPH	10	µM	isomenthone/menthone + nadph -> neomenthone, Km: >1000, pH 9.3, temp 30 c, from Mentha piperita, expressed in E.coli.	Davis2005^[1]
Km	Forward	NADPH	11	µM	isomenthone/menthone + nadph -> neomenthone, Km: >1000, pH 9.3, temp 30 c, from Mentha piperita, expressed in E.coli.	Davis2005^[1]
Km	Forward	NADPH	9	µM	isomenthone/menthone + nadph -> neomenthone, Km: >1000, pH 9.3, temp 30 c, from Mentha piperita, expressed in E.coli.	Davis2005^[1]
Km	Forward	menthone	22	µM	menthone -> neomenthol, pH 7.5, 30C, from Mentha piperita, expressed in E. coli	Kjonaas1982^[2]
Km	Forward	NADPH	20	µM	menthone -> neomenthol, pH 7.5, 30C, from Mentha piperita, expressed in E. coli	Kjonaas1982^[2]

Parameter uncertainty

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

**Values and weights of Km for menthone (MNMR)**
Parameter	Direction	Substrate	Value	Unit	Weight	weight counter	Probability distribution	Parameter details
Km	Forward	menthone	5.7	µM	512	512		Mode: 56.4
Km	Forward	menthone	7.1	µM	512	1024		Value Range: 5.3015 - 600.0114 μM
Km	Forward	menthone	8.5	µM	512	1536		CI factor: 10.6385
Km	Forward	menthone	22	µM	256	1792		μ: 4.8586
Km	Forward	menthone	90.8	µM	512	2304		σ: 0.9089
Km	Forward	menthone	96	µM	512	2816
Km	Forward	menthone	101.2	µM	512	3328
Km	Forward	menthone	596	µM	128	3456
Km	Forward	menthone	674	µM	128	3584
Km	Forward	menthone	752	µM	128	3712

**Values and weights of Km for isomenthone (MNMR)**
Parameter	Direction	Substrate	Value	Unit	Weight	weight counter	Probability distribution	Parameter details
Km	Forward	isomenthone	169.7	µM	512	512		Mode: 194.9
Km	Forward	isomenthone	186.5	µM	512	1024		Value Range: 62.0766 - 611.9211 μM
Km	Forward	isomenthone	203.3	µM	512	1536		μ: 5.5346
Km	Forward	isomenthone	1000	µM	128	1664		σ: 0.512

**Values and weights of Km for NADPH (MNMR)**
Parameter	Direction	Substrate	Value	Unit	Weight	weight counter	Probability distribution	Parameter details
Km	Forward	NADPH	9	µM	128	128		Mode: 11
Km	Forward	NADPH	10	µM	128	256		Value Range: 7.5342 - 16.06 μM
Km	Forward	NADPH	11	µM	128	384		μ: 2.4325
Km	Forward	NADPH	20	µM	256	640		σ: 0.186

**Values and weights of Km for Neomenthol (MNMR)**
Parameter	Direction	Substrate	Value	Unit	Weight	weight counter	Probability distribution	Parameter details
Km	Reverse	neomenthol	205	µM	512	512		Mode: 305
Km	Reverse	neomenthol	305	µM	512	1024		Value Range: 216.6444 - 429.3903 μM
Km	Reverse	neomenthol	405	µM	512	1536		μ: 5.74875; σ: 0.16865

**Values and weights of Kcat for menthone (MNMR)**
Parameter	Direction	Substrate	Value	Unit	Weight	weight counter	Probability distribution	Parameter details
Kcat	Forward	menthone	0.6	1/s	512	512		Mode: 0.87
Kcat	Forward	menthone	0.87	1/s	512	1024		Value Range: 0.6971-1.0858 s^-1
Kcat	Forward	menthone	0.91	1/s	512	1536		μ: -0.12713 ; σ: 0.1101

**Values and weights of Kcat for isomenthone (MNMR)**
Parameter	Direction	Substrate	Value	Unit	Weight	weight counter	Probability distribution	Parameter details
Kcat	Forward	isomenthone	0.42	1/s	512	512		Mode: 0.44
Kcat	Forward	isomenthone	0.44	1/s	512	1024		Value Range: 0.4204-0.4605 s^-1
Kcat	Forward	isomenthone	0.46	1/s	512	1536		μ: -0.82046 ; σ: 0.02273

Detailed description of kinetic values obtained from literature

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

Simulations

References

↑ ^1.0 ^1.1 ^1.2 ^1.3 ^1.4 ^1.5 ^1.6 ^1.7 Davis, E.M. et. al. (2005). "Monoterpene metabolism. Cloning, Expression, and Characterization of Menthone Reductases from Peppermint.", Vol 137, pp. 873-881 Cite error: Invalid <ref> tag; name "Davis2005" defined multiple times with different content
↑ ^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. Cite error: Invalid <ref> tag; name "Kjonaas1982" defined multiple times with different content
↑ 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 Toogood, H.S et. al. (2015). "Enzymatic menthol production: One-pot approach using engineered Escherichia coli", ACS Synth Biol, Vol. 4, pp. 1112-1123
↑ ^5.0 ^5.1 ^5.2 ^5.3 ^5.4 ^5.5 ^5.6 ^5.7 Ryden, A. et. al. (2010). "Molecular cloning and characterization of a broad substrate terpenoid oxidoreductase from Artemisia annua", Plant Cell Physiol., Vol. 51(7), pp. 1219-1228

[Davis2005-1] 1.0 ^1.1 ^1.2 ^1.3 ^1.4 ^1.5 ^1.6 ^1.7 Davis, E.M. et. al. (2005). "Monoterpene metabolism. Cloning, Expression, and Characterization of Menthone Reductases from Peppermint.", Vol 137, pp. 873-881 Cite error: Invalid <ref> tag; name "Davis2005" defined multiple times with different content

[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. Cite error: Invalid <ref> tag; name "Kjonaas1982" defined multiple times with different content

[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 Toogood, H.S et. al. (2015). "Enzymatic menthol production: One-pot approach using engineered Escherichia coli", ACS Synth Biol, Vol. 4, pp. 1112-1123

[Ryden2010-5] 5.0 ^5.1 ^5.2 ^5.3 ^5.4 ^5.5 ^5.6 ^5.7 Ryden, A. et. al. (2010). "Molecular cloning and characterization of a broad substrate terpenoid oxidoreductase from Artemisia annua", Plant Cell Physiol., Vol. 51(7), pp. 1219-1228

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Menthone:Neomenthol reductase (MNMR)

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 for menthone, isomenthone, neomenthol, neoisomenthol and NADPH

Parameter uncertainty

Detailed description of kinetic values obtained from literature

Simulations

References

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