Information on EC 1.14.11.56 - L-proline cis-4-hydroxylase

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The enzyme appears in viruses and cellular organisms

EC NUMBER
COMMENTARY hide
1.14.11.56
-
RECOMMENDED NAME
GeneOntology No.
L-proline cis-4-hydroxylase
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REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
L-proline + 2-oxoglutarate + O2 = cis-4-hydroxy-L-proline + succinate + CO2
show the reaction diagram
SYSTEMATIC NAME
IUBMB Comments
L-proline,2-oxoglutarate:oxygen oxidoreductase (cis-4-hydroxylating)
Requires Fe2+ and ascorbate. The enzyme, isolated from Rhizobium species, only produces cis-4-hydroxy-L-proline (cf. EC 1.14.11.57, L-proline trans-4-hydroxylase).
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
evolution
physiological function
additional information
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
L-pipecolate + 2-oxoglutarate + O2
cis-5-hydroxypipecolate + cis-3-hydroxypipecolate + succinate + CO2
show the reaction diagram
L-pipecolic acid + 2-oxoglutarate + O2
cis-5-hydroxy-L-pipecolic acid + cis-3-hydroxy-L-pipecolic acid + succinate + CO2
show the reaction diagram
L-proline + 2-oxoglutarate + O2
cis-4-hydroxy-L-proline + succinate + CO2
show the reaction diagram
NATURAL SUBSTRATES
NATURAL PRODUCTS
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
L-pipecolate + 2-oxoglutarate + O2
cis-5-hydroxypipecolate + cis-3-hydroxypipecolate + succinate + CO2
show the reaction diagram
L-proline + 2-oxoglutarate + O2
cis-4-hydroxy-L-proline + succinate + CO2
show the reaction diagram
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
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
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
1,10-phenanthroline
diethyldicarbonate
Fe3+
-
2 mM, 68% residual activity
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.25 - 0.3
2-oxoglutarate
0.54
L-proline
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
24 - 25
L-proline
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
44 - 46
L-proline
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.1
mutant V97F, product cis-3-hydroxy-L-pipecolic acid, pH 7.2, 30C
0.29
wild-type, product cis-5-hydroxy-L-pipecolic acid, pH 7.2, 30C
0.34
wild-type, product cis-3-hydroxy-L-pipecolic acid, pH 7.2, 30C
0.79
mutant V97F, product cis-5-hydroxy-L-pipecolic acid, pH 7.2, 30C
684
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pH 7.5, 25C
707
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pH 7.5, 25C
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
PDB
SCOP
CATH
ORGANISM
UNIPROT
Mesorhizobium japonicum (strain LMG 29417 / CECT 9101 / MAFF 303099)
Mesorhizobium japonicum (strain LMG 29417 / CECT 9101 / MAFF 303099)
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
homology modeling based in the Rhizobium loti structure; 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
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 15C, 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
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
recombinant Strep-tagged wild-type enzyme from Escherichia coli strain W3110 extract by ultracentrifugation, affinity and anion exchange chrmatography, and gel filtration
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expression in Escherichia coli
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expression in Escherichia coli; expression in Escherichia coli
recombinant expression of Strep-tagged wild-type enzyme in Escherichia coli strain W3110
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recombinant expression of wild-type and mutant enzymes in Escherichia coli strain W3110
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
V95A
no increase in hydroxylation activity nor any improvement in the cis-5/cis-3 product ratio
V95A/V97A
no increase in hydroxylation activity nor any improvement in the cis-5/cis-3 product ratio
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
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
V97C
4fold increase in production of cis-5-hydroxy-L-pipecolic acid, ration of -cis/5cis products is similar to wild-type
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
V97F/V95W
significantly improved regioselectivity toward cis-5-hydroxy-L-pipecolic acid
V97F/V95W/E114G
significantly improved regioselectivity toward cis-5-hydroxy-L-pipecolic acid and high productivity
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
V95A
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no increase in hydroxylation activity nor any improvement in the cis-5/cis-3 product ratio
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V95A/V97A
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no increase in hydroxylation activity nor any improvement in the cis-5/cis-3 product ratio
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V97A
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2.5fold increase in production of cis-5-hydroxy-L-pipecolic acid, ratio of cis-5/cis-3 products is similar to wild-type
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V97C
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4fold increase in production of cis-5-hydroxy-L-pipecolic acid, ration of -cis/5cis products is similar to wild-type
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V97F
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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
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V95A
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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
V95A/V97A
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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
V97A
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site-directed mutagenesis, the mutant shows increased activity and production of cis-4-hydroxy-L-proline
V97C
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site-directed mutagenesis
V97F
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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
V97F/V95W
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site-directed mutagenesis, the mutant shows improved regioselectivity and increased activity of hydroxylation
V97F/V95W/E114G
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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
V97Y
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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
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
synthesis
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coexpression of L-proline cis-4-hydroxylase and N-acetyltransferase Mpr1 from Saccharomyces cerevisiae converting cis-4-hydroxy-L-proline into N-acetyl cis-4-hydroxy-L-proline in Escherichia coli. M9 medium containing L-proline produces more N-acetyl cis-4-hydroxy-L-proline than LB medium containing L-proline. The addition of NaCl and L-ascorbate results in a 2fold increase in N-acetyl cis-4-hydroxy-L-proline production in the L-proline-containing M9 medium