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
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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
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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).
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
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-proline + 2-oxoglutarate + O2
cis-4-hydroxy-L-proline + succinate + CO2
show the reaction diagram
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
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, 30°C
0.29
wild-type, product cis-5-hydroxy-L-pipecolic acid, pH 7.2, 30°C
0.34
wild-type, product cis-3-hydroxy-L-pipecolic acid, pH 7.2, 30°C
0.79
mutant V97F, product cis-5-hydroxy-L-pipecolic acid, pH 7.2, 30°C
684
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pH 7.5, 25°C
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
PDB
SCOP
CATH
ORGANISM
UNIPROT
Rhizobium loti (strain MAFF303099)
Rhizobium loti (strain MAFF303099)
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
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expression in Escherichia coli
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expression in Escherichia coli; expression in Escherichia coli
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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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