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
recombinant expression of Strep-tagged wild-type enzyme in Escherichia coli strain W3110
Mesorhizobium loti
recombinant expression of wild-type and mutant enzymes in Escherichia coli strain W3110
Sinorhizobium meliloti
Crystallization (Commentary)
Crystallization (Commentary)
Organism
purified recombinant enzyme MlP4H in complex with Co2+, 2-oxoglutarate and L-Pro or L-Pip, the MlP4H protein used for crystallization includes an extra (Met)-Ser-Ala-Trp-Ser-His-Pro-Gln-Phe-Gly-Lys-Gly-Ala strep-tag II peptide at its N-terminus, and the original start codon of the wild-type is replaced by the underlined alanine, followed by the second codon of the wild-type. Crystallization of L-Pro complex crystals by sitting-drop vapor diffusion method mixing 0.001 ml of 28 mg/ml protein solution containing 2 mM CoCl2, 10 mM 2-oxoglutarate, and 20 mM L-Pro with reservoir solution containing 0.1 M bis-Tris propane, pH 8.5, 0.2 M sodium malonate, and 25% v/v PEG 3350, or of L-Pip complex crystals by sitting drop vapour diffusion method mixing 0.001 ml of 28 mg/ml protein solution containing 2 mM CoCl2, 10 mM ?2-oxoglutarate, and 20 mM L-Pip with 0.001 ml of reservoir solution containing 0.1 M CAPS, pH 10.5, 0.1 M lithium sulfate, and 1.8 M ammonium sulfate, all at 15°C, X-ray diffraction structure determination and analysis at 1.3-2.8 A resolution, by single-wavelength dispersion method with the bound Co2+ at the active site used as the anomalous scatter or by molecular replacement using the first L-Pro-bound structure as a search model
Mesorhizobium loti
Engineering
Protein Variants
Commentary
Organism
V95A
site-directed mutagenesis, the mutant does not show any increase in hydroxylation activity nor any improvement in the cis-5/cis-3 ratio compared to the wild-type enzyme
Sinorhizobium meliloti
V95A/V97A
site-directed mutagenesis, the mutant does not show any increase in hydroxylation activity nor any improvement in the cis-5/cis-3 ratio compared to the wild-type enzyme
Sinorhizobium meliloti
V97A
site-directed mutagenesis, the mutant shows increased activity and production of cis-4-hydroxy-L-proline
Sinorhizobium meliloti
V97C
site-directed mutagenesis
Sinorhizobium meliloti
V97F
site-directed mutagenesis, the mutant shows improved regioselectivity of hydroxylation, the cis-5/cis-3 ratio improves from 1.4 for the wild-type enzyme to 5.3 for the mutant, the increase in activity is similar compared to mutant V97A, the V97F mutant demonstrates higher selectivity of C5-hydroxylation
Sinorhizobium meliloti
V97F/V95W
site-directed mutagenesis, the mutant shows improved regioselectivity and increased activity of hydroxylation
Sinorhizobium meliloti
V97F/V95W/E114G
site-directed mutagenesis, protein engineering of L-proline cis-4-hydroxylase based on the X-ray crystal structure leading to refined regio- and stereoselective hydroxylation of L-pipecolic acid, the engineered mutant enzyme produces 96% cis-5-hydroxypipecolate and 4% cis-3-hydroxypipecolate while the wild-type produces 60% cis-5-hydroxypipecolate and 40% cis-3-hydroxypipecolate. A structure homology model of the SmP4H triple mutant V97F/V95W/E114G is constructed based on the MlP4H crystal structure. addition of the E114G mutation improves the activity approximately 2fold compared to double mutant V97F/V95W. The triple mutant shows the highest growth and productivity of cis-5-hydroxy-L-pipecolate in a regioselective manner
Sinorhizobium meliloti
V97Y
site-directed mutagenesis, the mutant shows improved regioselectivity of hydroxylation, the cis-5/cis-3 ratio improves from 1.4 for the wild-type enzyme to 9.0 for the mutant, the increase in activity is decreased compared to mutant V97F
Sinorhizobium meliloti
Metals/Ions
Metals/Ions
Commentary
Organism
Structure
Co2+
exogenous Co2+ is coordinated by residues H106, H154, and D108. Co2+ is a mimic of the catalytic metal center because of its stability under aerobic conditions and its enzymatic inactivity under anaerobic conditions. The cis-face of the C4 carbon of L-Pro is properly oriented toward Co2+, which in nature exists as FeIV=O during the hydroxylation reaction, to generate the enantiopure cis-4-hydroxyproline
Mesorhizobium loti
Fe2+
dependent on
Mesorhizobium loti
Fe2+
dependent on
Sinorhizobium meliloti
Natural Substrates/ Products (Substrates)
Natural Substrates
Organism
Commentary (Nat. Sub.)
Natural Products
Commentary (Nat. Pro.)
Organism (Nat. Pro.)
Reversibility
ID
L-pipecolate + 2-oxoglutarate + O2
Mesorhizobium loti
-
cis-5-hydroxypipecolate + cis-3-hydroxypipecolate + succinate + CO2
-
-
?
L-pipecolate + 2-oxoglutarate + O2
Sinorhizobium meliloti
-
cis-5-hydroxypipecolate + cis-3-hydroxypipecolate + succinate + CO2
-
-
?
L-proline + 2-oxoglutarate + O2
Mesorhizobium loti
-
cis-4-hydroxy-L-proline + succinate + CO2
-
-
?
L-proline + 2-oxoglutarate + O2
Sinorhizobium meliloti
-
cis-4-hydroxy-L-proline + succinate + CO2
-
-
?
Organism
Organism
UniProt
Commentary
Textmining
Mesorhizobium loti
Q989T9
-
-
Sinorhizobium meliloti
Q92LF6
-
-
Purification (Commentary)
Purification (Commentary)
Organism
recombinant Strep-tagged wild-type enzyme from Escherichia coli strain W3110 extract by ultracentrifugation, affinity and anion exchange chrmatography, and gel filtration
Mesorhizobium loti
Reaction
Reaction
Commentary
Organism
Reaction ID
L-proline + 2-oxoglutarate + O2 = cis-4-hydroxy-L-proline + succinate + CO2
proposed catalytic mechanism of cis-P4H with L-proline and L-pipecolate as substrates with 2-oxoglutarate and O2, overview
Mesorhizobium loti
L-proline + 2-oxoglutarate + O2 = cis-4-hydroxy-L-proline + succinate + CO2
proposed catalytic mechanism of cis-P4H with L-proline and L-pipecolate as substrates with 2-oxoglutarate and O2, overview
Sinorhizobium meliloti
Substrates and Products (Substrate)
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
Substrate Product ID
L-pipecolate + 2-oxoglutarate + O2
-
743931
Mesorhizobium loti
cis-5-hydroxypipecolate + cis-3-hydroxypipecolate + succinate + CO2
-
-
-
?
L-pipecolate + 2-oxoglutarate + O2
-
743931
Sinorhizobium meliloti
cis-5-hydroxypipecolate + cis-3-hydroxypipecolate + succinate + CO2
-
-
-
?
L-proline + 2-oxoglutarate + O2
-
743931
Mesorhizobium loti
cis-4-hydroxy-L-proline + succinate + CO2
-
-
-
?
L-proline + 2-oxoglutarate + O2
-
743931
Sinorhizobium meliloti
cis-4-hydroxy-L-proline + succinate + CO2
-
-
-
?
Synonyms
Synonyms
Commentary
Organism
cis-P4H
-
Mesorhizobium loti
cis-P4H
-
Sinorhizobium meliloti
MlP4H
-
Mesorhizobium loti
SmP4H
-
Sinorhizobium meliloti
Temperature Optimum [°C]
Temperature Optimum [°C]
Temperature Optimum Maximum [°C]
Commentary
Organism
30
-
assay at
Mesorhizobium loti
30
-
assay at
Sinorhizobium meliloti
pH Optimum
pH Optimum Minimum
pH Optimum Maximum
Commentary
Organism
7.2
-
assay at
Mesorhizobium loti
7.2
-
assay at
Sinorhizobium meliloti
Cloned(Commentary) (protein specific)
Commentary
Organism
recombinant expression of Strep-tagged wild-type enzyme in Escherichia coli strain W3110
Mesorhizobium loti
recombinant expression of wild-type and mutant enzymes in Escherichia coli strain W3110
Sinorhizobium meliloti
Crystallization (Commentary) (protein specific)
Crystallization
Organism
purified recombinant enzyme MlP4H in complex with Co2+, 2-oxoglutarate and L-Pro or L-Pip, the MlP4H protein used for crystallization includes an extra (Met)-Ser-Ala-Trp-Ser-His-Pro-Gln-Phe-Gly-Lys-Gly-Ala strep-tag II peptide at its N-terminus, and the original start codon of the wild-type is replaced by the underlined alanine, followed by the second codon of the wild-type. Crystallization of L-Pro complex crystals by sitting-drop vapor diffusion method mixing 0.001 ml of 28 mg/ml protein solution containing 2 mM CoCl2, 10 mM 2-oxoglutarate, and 20 mM L-Pro with reservoir solution containing 0.1 M bis-Tris propane, pH 8.5, 0.2 M sodium malonate, and 25% v/v PEG 3350, or of L-Pip complex crystals by sitting drop vapour diffusion method mixing 0.001 ml of 28 mg/ml protein solution containing 2 mM CoCl2, 10 mM ?2-oxoglutarate, and 20 mM L-Pip with 0.001 ml of reservoir solution containing 0.1 M CAPS, pH 10.5, 0.1 M lithium sulfate, and 1.8 M ammonium sulfate, all at 15°C, X-ray diffraction structure determination and analysis at 1.3-2.8 A resolution, by single-wavelength dispersion method with the bound Co2+ at the active site used as the anomalous scatter or by molecular replacement using the first L-Pro-bound structure as a search model
Mesorhizobium loti
Engineering (protein specific)
Protein Variants
Commentary
Organism
V95A
site-directed mutagenesis, the mutant does not show any increase in hydroxylation activity nor any improvement in the cis-5/cis-3 ratio compared to the wild-type enzyme
Sinorhizobium meliloti
V95A/V97A
site-directed mutagenesis, the mutant does not show any increase in hydroxylation activity nor any improvement in the cis-5/cis-3 ratio compared to the wild-type enzyme
Sinorhizobium meliloti
V97A
site-directed mutagenesis, the mutant shows increased activity and production of cis-4-hydroxy-L-proline
Sinorhizobium meliloti
V97C
site-directed mutagenesis
Sinorhizobium meliloti
V97F
site-directed mutagenesis, the mutant shows improved regioselectivity of hydroxylation, the cis-5/cis-3 ratio improves from 1.4 for the wild-type enzyme to 5.3 for the mutant, the increase in activity is similar compared to mutant V97A, the V97F mutant demonstrates higher selectivity of C5-hydroxylation
Sinorhizobium meliloti
V97F/V95W
site-directed mutagenesis, the mutant shows improved regioselectivity and increased activity of hydroxylation
Sinorhizobium meliloti
V97F/V95W/E114G
site-directed mutagenesis, protein engineering of L-proline cis-4-hydroxylase based on the X-ray crystal structure leading to refined regio- and stereoselective hydroxylation of L-pipecolic acid, the engineered mutant enzyme produces 96% cis-5-hydroxypipecolate and 4% cis-3-hydroxypipecolate while the wild-type produces 60% cis-5-hydroxypipecolate and 40% cis-3-hydroxypipecolate. A structure homology model of the SmP4H triple mutant V97F/V95W/E114G is constructed based on the MlP4H crystal structure. addition of the E114G mutation improves the activity approximately 2fold compared to double mutant V97F/V95W. The triple mutant shows the highest growth and productivity of cis-5-hydroxy-L-pipecolate in a regioselective manner
Sinorhizobium meliloti
V97Y
site-directed mutagenesis, the mutant shows improved regioselectivity of hydroxylation, the cis-5/cis-3 ratio improves from 1.4 for the wild-type enzyme to 9.0 for the mutant, the increase in activity is decreased compared to mutant V97F
Sinorhizobium meliloti
Metals/Ions (protein specific)
Metals/Ions
Commentary
Organism
Structure
Co2+
exogenous Co2+ is coordinated by residues H106, H154, and D108. Co2+ is a mimic of the catalytic metal center because of its stability under aerobic conditions and its enzymatic inactivity under anaerobic conditions. The cis-face of the C4 carbon of L-Pro is properly oriented toward Co2+, which in nature exists as FeIV=O during the hydroxylation reaction, to generate the enantiopure cis-4-hydroxyproline
Mesorhizobium loti
Fe2+
dependent on
Mesorhizobium loti
Fe2+
dependent on
Sinorhizobium meliloti
Natural Substrates/ Products (Substrates) (protein specific)
Natural Substrates
Organism
Commentary (Nat. Sub.)
Natural Products
Commentary (Nat. Pro.)
Organism (Nat. Pro.)
Reversibility
ID
L-pipecolate + 2-oxoglutarate + O2
Mesorhizobium loti
-
cis-5-hydroxypipecolate + cis-3-hydroxypipecolate + succinate + CO2
-
-
?
L-pipecolate + 2-oxoglutarate + O2
Sinorhizobium meliloti
-
cis-5-hydroxypipecolate + cis-3-hydroxypipecolate + succinate + CO2
-
-
?
L-proline + 2-oxoglutarate + O2
Mesorhizobium loti
-
cis-4-hydroxy-L-proline + succinate + CO2
-
-
?
L-proline + 2-oxoglutarate + O2
Sinorhizobium meliloti
-
cis-4-hydroxy-L-proline + succinate + CO2
-
-
?
Purification (Commentary) (protein specific)
Commentary
Organism
recombinant Strep-tagged wild-type enzyme from Escherichia coli strain W3110 extract by ultracentrifugation, affinity and anion exchange chrmatography, and gel filtration
Mesorhizobium loti
Substrates and Products (Substrate) (protein specific)
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
ID
L-pipecolate + 2-oxoglutarate + O2
-
743931
Mesorhizobium loti
cis-5-hydroxypipecolate + cis-3-hydroxypipecolate + succinate + CO2
-
-
-
?
L-pipecolate + 2-oxoglutarate + O2
-
743931
Sinorhizobium meliloti
cis-5-hydroxypipecolate + cis-3-hydroxypipecolate + succinate + CO2
-
-
-
?
L-proline + 2-oxoglutarate + O2
-
743931
Mesorhizobium loti
cis-4-hydroxy-L-proline + succinate + CO2
-
-
-
?
L-proline + 2-oxoglutarate + O2
-
743931
Sinorhizobium meliloti
cis-4-hydroxy-L-proline + succinate + CO2
-
-
-
?
Temperature Optimum [°C] (protein specific)
Temperature Optimum [°C]
Temperature Optimum Maximum [°C]
Commentary
Organism
30
-
assay at
Mesorhizobium loti
30
-
assay at
Sinorhizobium meliloti
pH Optimum (protein specific)
pH Optimum Minimum
pH Optimum Maximum
Commentary
Organism
7.2
-
assay at
Mesorhizobium loti
7.2
-
assay at
Sinorhizobium meliloti
General Information
General Information
Commentary
Organism
evolution
proline hydroxylases are representative members of the nonheme Fe2+/2-oxoglutarate-dependent dioxygenase family
Mesorhizobium loti
evolution
proline hydroxylases are representative members of the nonheme Fe2+/2-oxoglutarate-dependent dioxygenase family
Sinorhizobium meliloti
additional information
I95, I97, and E114 are active site residues, the active site was composed of a distorted jelly roll beta-sheet core, which is sandwiched by the N-terminal and C-terminal alpha-helical domains
Mesorhizobium loti
additional information
V95, V97, and G114 are active site residues, a structure homology model of the SmP4H triple mutant V97F/V95W/E114G is constructed based on the MlP4H crystal structure
Sinorhizobium meliloti
physiological function
the enzyme catalyze the hydroxylation of L-proline, generating cis-4-hydroxy-L-proline, as well as the hydroxylation of L-pipecolic acid (L-Pip), generating two regioisomers, cis-5-hydroxypipecolate and cis-3-hydroxypipecolate in a 6:4 ratio
Mesorhizobium loti
physiological function
the enzyme catalyze the hydroxylation of L-proline, generating cis-4-hydroxy-L-proline, as well as the hydroxylation of L-pipecolic acid (L-Pip), generating two regioisomers, cis-5-hydroxypipecolate and cis-3-hydroxypipecolate in a 6:4 ratio
Sinorhizobium meliloti
General Information (protein specific)
General Information
Commentary
Organism
evolution
proline hydroxylases are representative members of the nonheme Fe2+/2-oxoglutarate-dependent dioxygenase family
Mesorhizobium loti
evolution
proline hydroxylases are representative members of the nonheme Fe2+/2-oxoglutarate-dependent dioxygenase family
Sinorhizobium meliloti
additional information
I95, I97, and E114 are active site residues, the active site was composed of a distorted jelly roll beta-sheet core, which is sandwiched by the N-terminal and C-terminal alpha-helical domains
Mesorhizobium loti
additional information
V95, V97, and G114 are active site residues, a structure homology model of the SmP4H triple mutant V97F/V95W/E114G is constructed based on the MlP4H crystal structure
Sinorhizobium meliloti
physiological function
the enzyme catalyze the hydroxylation of L-proline, generating cis-4-hydroxy-L-proline, as well as the hydroxylation of L-pipecolic acid (L-Pip), generating two regioisomers, cis-5-hydroxypipecolate and cis-3-hydroxypipecolate in a 6:4 ratio
Mesorhizobium loti
physiological function
the enzyme catalyze the hydroxylation of L-proline, generating cis-4-hydroxy-L-proline, as well as the hydroxylation of L-pipecolic acid (L-Pip), generating two regioisomers, cis-5-hydroxypipecolate and cis-3-hydroxypipecolate in a 6:4 ratio
Sinorhizobium meliloti
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
-
-
2
2
9
-
-
-
-
2
-
-
-
7
-
-
-
-
-
-
4
-
8
-
4
-
-
-
-
-
-
-
-
-
-
-
-
-
2
-
2
9
-
-
-
-
-
-
2
-
-
-
-
-
-
-
-
4
-
8
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
743931
Koketsu
Refined regio- and stereosele ...
Sinorhizobium meliloti, Mesorhizobium loti
ACS Synth. Biol.
4
383-392
2015
-
-
2
1
8
-
-
-
-
3
-
4
-
4
-
-
1
2
-
-
-
-
4
-
4
2
-
-
-
2
-
-
-
-
-
-
-
-
2
-
1
8
-
-
-
-
-
-
3
-
4
-
-
-
1
-
-
-
-
4
-
2
-
-
-
2
-
-
-
-
6
6
-
-
-
726771
Bach
Microbial production of N-acet ...
Sinorhizobium meliloti
Appl. Microbiol. Biotechnol.
97
247-257
2013
-
1
1
-
-
-
-
-
-
-
-
-
-
7
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
1
1
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
696034
Hara
Characterization of novel 2-ox ...
Sinorhizobium meliloti, Mesorhizobium loti, Mesorhizobium loti MAFF303099
Biochem. Biophys. Res. Commun.
379
882-886
2009
-
-
-
-
-
-
15
4
-
2
-
3
-
4
-
-
-
-
-
-
2
-
3
-
2
2
-
-
2
2
-
-
-
-
-
-
-
-
-
-
-
-
-
-
15
-
4
-
2
-
3
-
-
-
-
-
-
2
-
3
-
2
-
-
2
2
-
-
-
-
-
-
-
2
2