BRENDA - Enzyme Database
show all sequences of 1.14.11.56

Refined regio- and stereoselective hydroxylation of L-pipecolic acid by protein engineering of L-proline cis-4-hydroxylase based on the X-ray crystal structure

Koketsu, K.; Shomura, Y.; Moriwaki, K.; Hayashi, M.; Mitsuhashi, S.; Hara, R.; Kino, K.; Higuchi, Y.; ACS Synth. Biol. 4, 383-392 (2015)

Data extracted from this reference:

Cloned(Commentary)
Cloned (Commentary)
Organism
expression in Escherichia coli
Mesorhizobium loti
Crystallization (Commentary)
Crystallization (Commentary)
Organism
homology modeling based in the Rhizobium loti structure
Mesorhizobium loti
in complex with in complex with Co2+, 2-oxoglutarate as cofactors, and L-proline or L-pipecolic acid. The active site is composed of a distorted jelly roll beta-sheet core, which is sandwiched by the N-terminal and C-terminal alpha-helical domains. Co2+ is coordinated by residues H106, H154, and D108
Mesorhizobium loti
Engineering
Protein Variants
Commentary
Organism
V95A
no increase in hydroxylation activity nor any improvement in the cis-5/cis-3 product ratio
Mesorhizobium loti
V95A/V97A
no increase in hydroxylation activity nor any improvement in the cis-5/cis-3 product ratio
Mesorhizobium loti
V95W
mutation significantly reduces the amount of cis-3-hydroxy-L-pipecolic acid (<5%) production compared with that of cis-5-hydroxy-L-pipecolic acid
Mesorhizobium loti
V97A
2.5fold increase in production of cis-5-hydroxy-L-pipecolic acid, ratio of cis-5/cis-3 products is similar to wild-type
Mesorhizobium loti
V97C
4fold increase in production of cis-5-hydroxy-L-pipecolic acid, ration of §-cis/5cis products is similar to wild-type
Mesorhizobium loti
V97F
the cis-5/cis-3 product ratio improves from 1.4 for the wild-type enzyme to 5.3, the total amount of product is similar to wild-type
Mesorhizobium loti
V97F/V95W
significantly improved regioselectivity toward cis-5-hydroxy-L-pipecolic acid
Mesorhizobium loti
V97F/V95W/E114G
significantly improved regioselectivity toward cis-5-hydroxy-L-pipecolic acid and high productivity
Mesorhizobium loti
V97Y
the cis-5/cis-3 product ratio improves from 1.4 for the wild-type enzyme to 9.0, the total amount of product decreases
Mesorhizobium loti
Metals/Ions
Metals/Ions
Commentary
Organism
Structure
Iron
-
Mesorhizobium loti
Organism
Organism
UniProt
Commentary
Textmining
Mesorhizobium loti
Q92LF6
-
-
Mesorhizobium loti
Q989T9
-
-
Mesorhizobium loti 1021
Q92LF6
-
-
Mesorhizobium loti MAFF303099
Q989T9
-
-
Specific Activity [micromol/min/mg]
Specific Activity Minimum [µmol/min/mg]
Specific Activity Maximum [µmol/min/mg]
Commentary
Organism
0.1
-
mutant V97F, product cis-3-hydroxy-L-pipecolic acid, pH 7.2, 30°C
Mesorhizobium loti
0.29
-
wild-type, product cis-5-hydroxy-L-pipecolic acid, pH 7.2, 30°C
Mesorhizobium loti
0.34
-
wild-type, product cis-3-hydroxy-L-pipecolic acid, pH 7.2, 30°C
Mesorhizobium loti
0.79
-
mutant V97F, product cis-5-hydroxy-L-pipecolic acid, pH 7.2, 30°C
Mesorhizobium loti
Substrates and Products (Substrate)
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
Substrate Product ID
L-pipecolic acid + 2-oxoglutarate + O2
-
739780
Mesorhizobium loti
cis-5-hydroxy-L-pipecolic acid + cis-3-hydroxy-L-pipecolic acid + succinate + CO2
the ratio between cis-5/cis-3 isomers is about 1:7
-
-
?
L-pipecolic acid + 2-oxoglutarate + O2
-
739780
Mesorhizobium loti
cis-5-hydroxy-L-pipecolic acid + cis-3-hydroxy-L-pipecolic acid + succinate + CO2
the ratio between cis-5/cis-3 isomers is about 2:1
-
-
?
L-pipecolic acid + 2-oxoglutarate + O2
-
739780
Mesorhizobium loti MAFF303099
cis-5-hydroxy-L-pipecolic acid + cis-3-hydroxy-L-pipecolic acid + succinate + CO2
the ratio between cis-5/cis-3 isomers is about 1:7
-
-
?
L-pipecolic acid + 2-oxoglutarate + O2
-
739780
Mesorhizobium loti 1021
cis-5-hydroxy-L-pipecolic acid + cis-3-hydroxy-L-pipecolic acid + succinate + CO2
the ratio between cis-5/cis-3 isomers is about 2:1
-
-
?
L-proline + 2-oxoglutarate + O2
-
739780
Mesorhizobium loti
cis-4-hydroxy-L-proline + succinate + CO2
-
-
-
?
L-proline + 2-oxoglutarate + O2
-
739780
Mesorhizobium loti MAFF303099
cis-4-hydroxy-L-proline + succinate + CO2
-
-
-
?
L-proline + 2-oxoglutarate + O2
-
739780
Mesorhizobium loti 1021
cis-4-hydroxy-L-proline + succinate + CO2
-
-
-
?
Synonyms
Synonyms
Commentary
Organism
cis-P4H
-
Mesorhizobium loti
mlr6283
-
Mesorhizobium loti
R03107
-
Mesorhizobium loti
Cloned(Commentary) (protein specific)
Commentary
Organism
expression in Escherichia coli
Mesorhizobium loti
Crystallization (Commentary) (protein specific)
Crystallization
Organism
homology modeling based in the Rhizobium loti structure
Mesorhizobium loti
in complex with in complex with Co2+, 2-oxoglutarate as cofactors, and L-proline or L-pipecolic acid. The active site is composed of a distorted jelly roll beta-sheet core, which is sandwiched by the N-terminal and C-terminal alpha-helical domains. Co2+ is coordinated by residues H106, H154, and D108
Mesorhizobium loti
Engineering (protein specific)
Protein Variants
Commentary
Organism
V95A
no increase in hydroxylation activity nor any improvement in the cis-5/cis-3 product ratio
Mesorhizobium loti
V95A/V97A
no increase in hydroxylation activity nor any improvement in the cis-5/cis-3 product ratio
Mesorhizobium loti
V95W
mutation significantly reduces the amount of cis-3-hydroxy-L-pipecolic acid (<5%) production compared with that of cis-5-hydroxy-L-pipecolic acid
Mesorhizobium loti
V97A
2.5fold increase in production of cis-5-hydroxy-L-pipecolic acid, ratio of cis-5/cis-3 products is similar to wild-type
Mesorhizobium loti
V97C
4fold increase in production of cis-5-hydroxy-L-pipecolic acid, ration of §-cis/5cis products is similar to wild-type
Mesorhizobium loti
V97F
the cis-5/cis-3 product ratio improves from 1.4 for the wild-type enzyme to 5.3, the total amount of product is similar to wild-type
Mesorhizobium loti
V97F/V95W
significantly improved regioselectivity toward cis-5-hydroxy-L-pipecolic acid
Mesorhizobium loti
V97F/V95W/E114G
significantly improved regioselectivity toward cis-5-hydroxy-L-pipecolic acid and high productivity
Mesorhizobium loti
V97Y
the cis-5/cis-3 product ratio improves from 1.4 for the wild-type enzyme to 9.0, the total amount of product decreases
Mesorhizobium loti
Metals/Ions (protein specific)
Metals/Ions
Commentary
Organism
Structure
Iron
-
Mesorhizobium loti
Specific Activity [micromol/min/mg] (protein specific)
Specific Activity Minimum [µmol/min/mg]
Specific Activity Maximum [µmol/min/mg]
Commentary
Organism
0.1
-
mutant V97F, product cis-3-hydroxy-L-pipecolic acid, pH 7.2, 30°C
Mesorhizobium loti
0.29
-
wild-type, product cis-5-hydroxy-L-pipecolic acid, pH 7.2, 30°C
Mesorhizobium loti
0.34
-
wild-type, product cis-3-hydroxy-L-pipecolic acid, pH 7.2, 30°C
Mesorhizobium loti
0.79
-
mutant V97F, product cis-5-hydroxy-L-pipecolic acid, pH 7.2, 30°C
Mesorhizobium loti
Substrates and Products (Substrate) (protein specific)
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
ID
L-pipecolic acid + 2-oxoglutarate + O2
-
739780
Mesorhizobium loti
cis-5-hydroxy-L-pipecolic acid + cis-3-hydroxy-L-pipecolic acid + succinate + CO2
the ratio between cis-5/cis-3 isomers is about 1:7
-
-
?
L-pipecolic acid + 2-oxoglutarate + O2
-
739780
Mesorhizobium loti
cis-5-hydroxy-L-pipecolic acid + cis-3-hydroxy-L-pipecolic acid + succinate + CO2
the ratio between cis-5/cis-3 isomers is about 2:1
-
-
?
L-pipecolic acid + 2-oxoglutarate + O2
-
739780
Mesorhizobium loti MAFF303099
cis-5-hydroxy-L-pipecolic acid + cis-3-hydroxy-L-pipecolic acid + succinate + CO2
the ratio between cis-5/cis-3 isomers is about 1:7
-
-
?
L-pipecolic acid + 2-oxoglutarate + O2
-
739780
Mesorhizobium loti 1021
cis-5-hydroxy-L-pipecolic acid + cis-3-hydroxy-L-pipecolic acid + succinate + CO2
the ratio between cis-5/cis-3 isomers is about 2:1
-
-
?
L-proline + 2-oxoglutarate + O2
-
739780
Mesorhizobium loti
cis-4-hydroxy-L-proline + succinate + CO2
-
-
-
?
L-proline + 2-oxoglutarate + O2
-
739780
Mesorhizobium loti MAFF303099
cis-4-hydroxy-L-proline + succinate + CO2
-
-
-
?
L-proline + 2-oxoglutarate + O2
-
739780
Mesorhizobium loti 1021
cis-4-hydroxy-L-proline + succinate + CO2
-
-
-
?
Other publictions for EC 1.14.11.56
No.
1st author
Pub Med
title
organims
journal
volume
pages
year
Activating Compound
Application
Cloned(Commentary)
Crystallization (Commentary)
Engineering
General Stability
Inhibitors
KM Value [mM]
Localization
Metals/Ions
Molecular Weight [Da]
Natural Substrates/ Products (Substrates)
Organic Solvent Stability
Organism
Oxidation Stability
Posttranslational Modification
Purification (Commentary)
Reaction
Renatured (Commentary)
Source Tissue
Specific Activity [micromol/min/mg]
Storage Stability
Substrates and Products (Substrate)
Subunits
Synonyms
Temperature Optimum [°C]
Temperature Range [°C]
Temperature Stability [°C]
Turnover Number [1/s]
pH Optimum
pH Range
pH Stability
Cofactor
Ki Value [mM]
pI Value
IC50 Value
Activating Compound (protein specific)
Application (protein specific)
Cloned(Commentary) (protein specific)
Cofactor (protein specific)
Crystallization (Commentary) (protein specific)
Engineering (protein specific)
General Stability (protein specific)
IC50 Value (protein specific)
Inhibitors (protein specific)
Ki Value [mM] (protein specific)
KM Value [mM] (protein specific)
Localization (protein specific)
Metals/Ions (protein specific)
Molecular Weight [Da] (protein specific)
Natural Substrates/ Products (Substrates) (protein specific)
Organic Solvent Stability (protein specific)
Oxidation Stability (protein specific)
Posttranslational Modification (protein specific)
Purification (Commentary) (protein specific)
Renatured (Commentary) (protein specific)
Source Tissue (protein specific)
Specific Activity [micromol/min/mg] (protein specific)
Storage Stability (protein specific)
Substrates and Products (Substrate) (protein specific)
Subunits (protein specific)
Temperature Optimum [°C] (protein specific)
Temperature Range [°C] (protein specific)
Temperature Stability [°C] (protein specific)
Turnover Number [1/s] (protein specific)
pH Optimum (protein specific)
pH Range (protein specific)
pH Stability (protein specific)
pI Value (protein specific)
Expression
General Information
General Information (protein specific)
Expression (protein specific)
KCat/KM [mM/s]
KCat/KM [mM/s] (protein specific)
739780
Koketsu
Refined regio- and stereoselec ...
Mesorhizobium loti, Mesorhizobium loti MAFF303099, Mesorhizobium loti 1021
ACS Synth. Biol.
4
383-392
2015
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2
2
9
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2
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7
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4
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8
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4
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2
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2
9
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2
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4
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8
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743931
Koketsu
Refined regio- and stereosele ...
Sinorhizobium meliloti, Mesorhizobium loti
ACS Synth. Biol.
4
383-392
2015
-
-
2
1
8
-
-
-
-
3
-
4
-
4
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1
2
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4
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4
2
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2
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2
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1
8
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3
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4
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1
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4
-
2
-
-
-
2
-
-
-
-
6
6
-
-
-
726771
Bach
Microbial production of N-acet ...
Sinorhizobium meliloti
Appl. Microbiol. Biotechnol.
97
247-257
2013
-
1
1
-
-
-
-
-
-
-
-
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7
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1
1
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696034
Hara
Characterization of novel 2-ox ...
Sinorhizobium meliloti, Mesorhizobium loti, Mesorhizobium loti MAFF303099
Biochem. Biophys. Res. Commun.
379
882-886
2009
-
-
-
-
-
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15
4
-
2
-
3
-
4
-
-
-
-
-
-
2
-
3
-
2
2
-
-
2
2
-
-
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-
-
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-
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-
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-
15
-
4
-
2
-
3
-
-
-
-
-
-
2
-
3
-
2
-
-
2
2
-
-
-
-
-
-
-
2
2