Information on EC 5.4.3.5 - D-ornithine 4,5-aminomutase

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The expected taxonomic range for this enzyme is: Bacteria

EC NUMBER
COMMENTARY
5.4.3.5
-
RECOMMENDED NAME
GeneOntology No.
D-ornithine 4,5-aminomutase
REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT
LITERATURE
D-ornithine = (2R,4S)-2,4-diaminopentanoate
show the reaction diagram
-
-
-
-
D-ornithine = (2R,4S)-2,4-diaminopentanoate
show the reaction diagram
catalytic mechanism, overview
-
D-ornithine = (2R,4S)-2,4-diaminopentanoate
show the reaction diagram
mechanism, overview, a gradual weakening of the electrostatic energy between the protein and the ribose leads to a progressive increase in the activation energy barrier for adenosylcobalamin Co-C bond homolysis, key role for the conserved polar glutamate residue in controlling the initial generation of radical species, mechanism, overview, a gradual weakening of the electrostatic energy between the protein and the ribose leads to a progressive increase in the activation energy barrier for adenosylcobalamin Co?C bond homolysis, key role for the conserved polar glutamate residue in controlling the initial generation of radical species
-
D-ornithine = (2R,4S)-2,4-diaminopentanoate
show the reaction diagram
radical-based catalysis mechanism, closed, active enzyme form modeling, overview
-
D-ornithine = (2R,4S)-2,4-diaminopentanoate
show the reaction diagram
radical-based catalysis that is initiated and propagated by the enzyme's adenosylcobalamin and pyridoxal 5'-phosphate cofactors. Following transaldimination, the Co-C bond of adenosylcobalamin undergoes homolytic rupture, generating a highly reactive carbon-centered 5'-deoxyadenosyl radical and cob(II)alamin. The 5'-deoxyadenosyl radical abstracts the C4 hydrogen atom from the D-ornithinyl-pyridoxal 5'-phosphate aldimine producing a substrate radical, which undergoes internal addition to the imine N to form an aziridylcarbinyl-pyridoxal 5'-phosphate radical adduct. Ring opening leads to formation of a product-like radical intermediate 3, which reabstracts a hydrogen atom from 5'-deoxyadenosine. Adenosylcobalamin is reformed with geminate recombination between the 5'-deoxyadenosyl radical and cob(II)alamin. Release of product from pyridoxal 5'-phosphate completes the catalytic cycle, radical-based catalysis that is initiated and propagated by the enzyme's adenosylcobalamin and pyridoxal 5'-phosphate cofactors. Following transaldimination, the Co-C bond of adenosylcobalamin undergoes homolytic rupture, generating a highly reactive carbon-centered 5'-deoxyadenosyl radical and cob(II)alamin. The 5'-deoxyadenosyl radical abstracts the C4 hydrogen atom from the D-ornithinylpyridoxal 5'-phosphate aldimine producing a substrate radical, which undergoes internal addition to the imine N to form an aziridylcarbinyl-pyridoxal 5'-phosphate radical adduct. Ring opening leads to formation of a product-like radical intermediate 3, which reabstracts a hydrogen atom from 5'-deoxyadenosine. Adenosylcobalamin is reformed with geminate recombination between the 5'-deoxyadenosyl radical and cob(II)alamin. Release of product from pyridoxal 5'-phosphate completes the catalytic cycle
-
D-ornithine = (2R,4S)-2,4-diaminopentanoate
show the reaction diagram
radical-based catalysis that is initiated and propagated by the enzyme's adenosylcobalamin and pyridoxal 5'-phosphate cofactors. Following transaldimination, the Co-C bond of adenosylcobalamin undergoes homolytic rupture, generating a highly reactive carbon-centered 5'-deoxyadenosyl radical and cob(II)alamin. The 5'-deoxyadenosyl radical abstracts the C4 hydrogen atom from the D-ornithinylpyridoxal 5'-phosphate aldimine producing a substrate radical, which undergoes internal addition to the imine N to form an aziridylcarbinyl-pyridoxal 5'-phosphate radical adduct. Ring opening leads to formation of a product-like radical intermediate 3, which reabstracts a hydrogen atom from 5'-deoxyadenosine. Adenosylcobalamin is reformed with geminate recombination between the 5'-deoxyadenosyl radical and cob(II)alamin. Release of product from pyridoxal 5'-phosphate completes the catalytic cycle
Clostridium sticklandii DSM S19
-
-
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
isomerization
-
-
-
-
PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
D-Arginine and D-ornithine metabolism
-
-
L-ornithine degradation II (Stickland reaction)
-
-
urea cycle
-
-
SYSTEMATIC NAME
IUBMB Comments
D-ornithine 4,5-aminomutase
A pyridoxal-phosphate protein that requires a cobamide coenzyme for activity.
SYNONYMS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
Aminomutase, D-ornithine 4,5-
-
-
-
-
CAS REGISTRY NUMBER
COMMENTARY
62213-30-3
-
ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
strain OhlLAs
-
-
Manually annotated by BRENDA team
Alkaliphilus oremlandii OhlLAs
strain OhlLAs
-
-
Manually annotated by BRENDA team
strain ATCC 12662
-
-
Manually annotated by BRENDA team
strain DSM 619
-
-
Manually annotated by BRENDA team
Clostridium sticklandii DSM S19
beta-subunit
UniProt
Manually annotated by BRENDA team
strain JW/NM-WN-LF
-
-
Manually annotated by BRENDA team
strain SJ95
-
-
Manually annotated by BRENDA team
strain SJ95
-
-
Manually annotated by BRENDA team
strain KPA171202
-
-
Manually annotated by BRENDA team
Thermoanaerobacter pseudethanolicus MB4
strain MB4
-
-
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
evolution
-
the enzyme belongs to the class III dAdoCbl-dependent isomerase family
metabolism
-
the enzyme catalyzes the second step in the oxidative breakdown of the amino acid, converting D-ornithine to 2,4-diaminopentanoic acid
metabolism
Clostridium sticklandii DSM S19
-
the enzyme catalyzes the second step in the oxidative breakdown of the amino acid, converting D-ornithine to 2,4-diaminopentanoic acid
-
metabolism
Thermoanaerobacter pseudethanolicus MB4, Alkaliphilus oremlandii OhlLAs
-
catalyzes step 2 in the ornithine fermentation pathway
-
additional information
-
going from the open, catalytically inactive form to the closed, catalytically active form, the Rossmann domain of the enzyme effectively approaches the active site as a rigid body. It undergoes a combination of a about 52 rotation and a 14 A translation to bring AdoCbl, initially positioned about 25 A away, into the active site cavity. This process is coupled to repositioning of the Ado moiety of adenosylcobalamin from the eastern conformation to the northern conformation. Combined quantum mechanics and molecular mechanics calculations further indicate that in the open form, the protein environment does not impact significantly on the Co-C bond homolytic rupture, rendering it unusually stable, and thus catalytically inactive
additional information
-
modeling of the closed conformation of the enzyme, domain motions, overview
SUBSTRATE
PRODUCT                      
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
LITERATURE
(Substrate)
COMMENTARY
(Product)
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
D-Orn
L(4S)-2,4-Diaminopentanoate
show the reaction diagram
-
r
-
-
D-Orn
?
show the reaction diagram
-
enzyme of ornithine fermentation
-
-
-
D-ornithine
(2R,4S)-2,4-diaminopentanoate
show the reaction diagram
-
-
-
-
?
D-ornithine
(2R,4S)-2,4-diaminopentanoate
show the reaction diagram
-
-
-
r
D-ornithine
(2R,4S)-2,4-diaminopentanoate
show the reaction diagram
-
-
-
-
r
D-ornithine
(2R,4S)-2,4-diaminopentanoate
show the reaction diagram
E3PY95, E3PY96
-
-
-
?
D-ornithine
(2R,4S)-2,4-diaminopentanoate
show the reaction diagram
E3PY95, E3PY96
-
-
-
?
D-ornithine
(2R,4S)-2,4-diaminopentanoate
show the reaction diagram
-
-
-
-
?
D-ornithine
(2R,4S)-2,4-diaminopentanoate
show the reaction diagram
-
second step in the oxidation pathway of L-ornithine
-
r
D-ornithine
(2R,4S)-2,4-diaminopentanoate
show the reaction diagram
E3PY95, E3PY96
binding of substrate to the enzyme leads to the formation of an electrostatic interaction between a conserved glutamate side chain and the adenosyl ribose of the adenosylcobalamin cofactor. Residue Glu338 is involved in adenosylcobalamin Co-C bond labilization and catalysis
-
-
?
D-ornithine
(2R,4S)-2,4-diaminopentanoate
show the reaction diagram
-
the 1,2-amino shift performed by the enzyme is considered energetically challenging as it involves breakage of chemically inert C-H and C-N bonds. OAM overcomes this thermodynamic barrier with radical-based catalysis that is initiated and propagated by the enzyme's adenosylcobalamin and pyridoxal 5'-phosphate cofactors, conformational change upon substrate binding, overview, the 1,2-amino shift performed by the enzyme is considered energetically challenging as it involves breakage of chemically inert CH and CN bonds. OAM overcomes this thermodynamic barrier with radical-based catalysis that is initiated and propagated by the enzyme's adenosylcobalamin and pyridoxal 5'-phosphate cofactors, conformational change upon substrate binding, overview
-
-
?
D-ornithine
(2R,4S)-2,4-diaminopentanoate
show the reaction diagram
-
the enzyme is highly specific for D-ornithine as a substrate, substrate binding structure, overview
-
-
r
D-ornithine
(2R,4S)-2,4-diaminopentanoate
show the reaction diagram
Clostridium sticklandii DSM S19
E3PY95
the 1,2-amino shift performed by the enzyme is considered energetically challenging as it involves breakage of chemically inert CH and CN bonds. OAM overcomes this thermodynamic barrier with radical-based catalysis that is initiated and propagated by the enzyme's adenosylcobalamin and pyridoxal 5'-phosphate cofactors, conformational change upon substrate binding, overview
-
-
?
D-ornithine
(2R,4S)-2,4-diaminopentanoate
show the reaction diagram
Thermoanaerobacter pseudethanolicus MB4, Alkaliphilus oremlandii OhlLAs
-
-
-
-
r
additional information
?
-
-
subunit OraS of the enzyme is capable of forming a complex with recombinant enzyme (KamDE) containing only E1 of lysine 5,6-aminomutase, EC 5.4.3.4, and restores its allosteric regulation by ATP
-
-
-
NATURAL SUBSTRATES
NATURAL PRODUCTS
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
LITERATURE
(Substrate)
COMMENTARY
(Product)
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
D-Orn
?
show the reaction diagram
-
enzyme of ornithine fermentation
-
-
-
D-ornithine
(2R,4S)-2,4-diaminopentanoate
show the reaction diagram
-
-
-
-
?
D-ornithine
(2R,4S)-2,4-diaminopentanoate
show the reaction diagram
-
-
-
-
r
D-ornithine
(2R,4S)-2,4-diaminopentanoate
show the reaction diagram
E3PY95, E3PY96
-
-
-
?
D-ornithine
(2R,4S)-2,4-diaminopentanoate
show the reaction diagram
E3PY95, E3PY96
-
-
-
?
D-ornithine
(2R,4S)-2,4-diaminopentanoate
show the reaction diagram
-
-
-
-
?
D-ornithine
(2R,4S)-2,4-diaminopentanoate
show the reaction diagram
-
second step in the oxidation pathway of L-ornithine
-
r
D-ornithine
(2R,4S)-2,4-diaminopentanoate
show the reaction diagram
Clostridium sticklandii DSM S19
E3PY95
-
-
-
?
D-ornithine
(2R,4S)-2,4-diaminopentanoate
show the reaction diagram
Thermoanaerobacter pseudethanolicus MB4, Alkaliphilus oremlandii OhlLAs
-
-
-
-
r
additional information
?
-
-
subunit OraS of the enzyme is capable of forming a complex with recombinant enzyme (KamDE) containing only E1 of lysine 5,6-aminomutase, EC 5.4.3.4, and restores its allosteric regulation by ATP
-
-
-
COFACTOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
5'-deoxyadenosylcobalamin
-
Km value 0.00043
5'-deoxyadenosylcobalamin
-
shows ability to produce highly reactive 5'-deoxyadenosyl radical in enzymatic environments
adenosylcobalamin
E3PY95, E3PY96
;
adenosylcobalamin
-
binding analysis with recombinant wild-type and mutant enzymes, overview; binding analysis with recombinant wild-type and mutant enzymes, overview
adenosylcobalamin
-
paramagnetic Co2+ metal center of the cob(II)alamin cofactor; paramagnetic Co2+ metal center of the cob(II)alamin cofactor
adenosylcobalamin
-
dependent on
pyridoxal 5'-phosphate
-
required, Km: 0.00036 mM
pyridoxal 5'-phosphate
-
Km value 0.0015
pyridoxal 5'-phosphate
-
dependent on
pyridoxal 5'-phosphate
E3PY95, E3PY96
;
pyridoxal 5'-phosphate
-
covalently bound via a Schiff base (imine) to Lys629; covalently bound via a Schiff base (imine) to Lys629
pyridoxal 5'-phosphate
-
stabilizes high-energy intermediates for performing challenging 1,2-amino rearrangements between adjacent carbons, binding site structure, overview
pyridoxal 5'-phosphate
-
-
vitamin B12
-
required
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
Co2+
-
in the paramagnetic Co2+ metal center of the cob(II)alamin cofactor; in the paramagnetic Co2+ metal center of the cob(II)alamin cofactor
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
2,4-diamino-n-butyric acid
-
competitive
-
2,4-diaminobutyrate
E3PY95, E3PY96
;
2,4-diaminobutyric acid
-
;
2,4-Diaminopentanoic acid
-
product inhibition above 0.7 mM
DL-2,4-diaminobutryic acid
-
-
DL-alpha-Lys
-
-
-
iodoacetate
-
-
L-alpha-Orn
-
-
potassium phosphate
-
-
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
mercaptan
-
keeps essential labile sulfhydryl groups reduced and protects from oxygen
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.44
D-Orn
-
coupled assay with C4 dehydrogenase
6.7
D-Orn
-
radiochemical assay
0.03
D-ornithine
-
recombinant His-tagged mutant E338A, pH 7.5, 30C; recombinant His-tagged mutant E338A, pH 7.5, 30C
0.043
D-ornithine
-
recombinant His-tagged mutant E338D, pH 7.5, 30C; recombinant His-tagged mutant E338D, pH 7.5, 30C
0.045
D-ornithine
-
-
0.061
D-ornithine
-
recombinant His-tagged mutant E338Q, pH 7.5, 30C; recombinant His-tagged mutant E338Q, pH 7.5, 30C
0.14
D-ornithine
-
recombinant mutant H225A, pH 8.5, 25C; recombinant mutant H225A, pH 8.5, 25C
0.19
D-ornithine
-
recombinant His-tagged wild-type enzyme, pH 7.5, 30C; recombinant His-tagged wild-type enzyme, pH 7.5, 30C
0.19
D-ornithine
-
recombinant wild-type, pH 8.5, 25C; recombinant wild-type, pH 8.5, 25C
0.2
D-ornithine
-
pH and temperature not specified in the publication
0.453
D-ornithine
-
recombinant mutant H225Q, pH 8.5, 25C; recombinant mutant H225Q, pH 8.5, 25C
additional information
additional information
-
pre-steady-state and stedy-state kinetics of wild-type and mutant enzymes; pre-steady-state and stedy-state kinetics of wild-type and mutant enzymes
-
additional information
additional information
-
Michaelis-Menten kinetics; Michaelis-Menten kinetics
-
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.032
D-ornithine
-
recombinant His-tagged mutant E338D, pH 7.5, 30C; recombinant His-tagged mutant E338D, pH 7.5, 30C
0.3
D-ornithine
-
recombinant mutant H225A, pH 8.5, 25C; recombinant mutant H225A, pH 8.5, 25C
1
D-ornithine
-
recombinant mutant H225Q, pH 8.5, 25C; recombinant mutant H225Q, pH 8.5, 25C
2.9
D-ornithine
-
recombinant His-tagged wild-type enzyme, pH 7.5, 30C; recombinant His-tagged wild-type enzyme, pH 7.5, 30C
2.9
D-ornithine
-
recombinant wild-type, pH 8.5, 25C; recombinant wild-type, pH 8.5, 25C
4.3
D-ornithine
-
recombinant His-tagged mutant E338Q, pH 7.5, 30C; recombinant His-tagged mutant E338Q, pH 7.5, 30C
6.3
D-ornithine
-
-
7.6
D-ornithine
-
recombinant His-tagged mutant E338A, pH 7.5, 30C; recombinant His-tagged mutant E338A, pH 7.5, 30C
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.07
D-ornithine
-
recombinant His-tagged mutant E338Q, pH 7.5, 30C; recombinant His-tagged mutant E338Q, pH 7.5, 30C
2550
0.25
D-ornithine
-
recombinant His-tagged mutant E338A, pH 7.5, 30C; recombinant His-tagged mutant E338A, pH 7.5, 30C
2550
0.75
D-ornithine
-
recombinant His-tagged mutant E338D, pH 7.5, 30C; recombinant His-tagged mutant E338D, pH 7.5, 30C
2550
2.2
D-ornithine
-
recombinant mutant H225A, pH 8.5, 25C; recombinant mutant H225A, pH 8.5, 25C; recombinant mutant H225Q, pH 8.5, 25C; recombinant mutant H225Q, pH 8.5, 25C
2550
15.2
D-ornithine
-
recombinant His-tagged wild-type enzyme, pH 7.5, 30C; recombinant His-tagged wild-type enzyme, pH 7.5, 30C
2550
15.2
D-ornithine
-
recombinant wild-type, pH 8.5, 25C; recombinant wild-type, pH 8.5, 25C
2550
15.2
D-ornithine
-
pH and temperature not specified in the publication
2550
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.096
2,4-diamino-n-butyric acid
-
-
-
0.0046
2,4-diaminobutyric acid
-
recombinant wild-type, pH 8.5, 25C
0.095
2,4-diaminobutyric acid
-
recombinant mutant H225A, pH 8.5, 25C
0.098
2,4-diaminobutyric acid
-
recombinant mutant H225Q, pH 8.5, 25C
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
7.5
-
assay at; assay at
8.5
-
assay at; assay at
pH RANGE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
7.4 - 9.7
-
half maximal activities at pH 7.4 and 9.7
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
25
-
assay at; assay at
30
-
assay at; assay at
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
23 - 49
-
half maximal activities at 23C and 49C
PDB
SCOP
CATH
ORGANISM
Clostridium sticklandii (strain ATCC 12662 / DSM 519 / JCM 1433 / NCIB 10654)
Clostridium sticklandii (strain ATCC 12662 / DSM 519 / JCM 1433 / NCIB 10654)
Clostridium sticklandii (strain ATCC 12662 / DSM 519 / JCM 1433 / NCIB 10654)
Clostridium sticklandii (strain ATCC 12662 / DSM 519 / JCM 1433 / NCIB 10654)
Clostridium sticklandii (strain ATCC 12662 / DSM 519 / JCM 1433 / NCIB 10654)
Clostridium sticklandii (strain ATCC 12662 / DSM 519 / JCM 1433 / NCIB 10654)
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
12800
E3PY95, E3PY96
OraS
704658
82900
E3PY95, E3PY96
OraE
704658
180000
-
gel electrophoresis, gel filtration, sucrose density gradient centrifugation
2074
201000
-
gel filtration
652199
210000
-
-
727644
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
dimer
-
2 * 95000-98000, SDS-PAGE
tetramer
-
alpha2beta2, 2 * 12800 + 2 * 82900, deduced from nucleotide sequence
tetramer
-
2 * 12800, subunit OraS + 2 * 82900, subunit OraE, alpha2beta2
tetramer
-
2 * 12800, subunit OraS + 2 * 90000, subunit OraE, alpha2beta2
tetramer
Clostridium sticklandii DSM S19
-
2 * 12800, subunit OraS + 2 * 82900, subunit OraE, alpha2beta2
-
heterotetramer
-
alpha2beta2, OraS, OraE, 2 * 12800 + 2 * 82900
additional information
-
protein KamDE comprised of the 30000 and 51000 Da subunits of the E1 component of D-alpha-lysine aminomutase is catalytically active in absence of the third 12800 kDa subunit, but ATP no longer has a regulatory effect on it. The S subunit of D-ornithine aminomutase, OraS, is capable of forming a complex with KamDE and restores the enzymes ATP-dependent allosteric regulation
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
crystal structure analysis
-
the structure of substrate-free ornithine 4,5-aminomutase is solved to a resolution of 2.0 A, furthermore the structures of the enzyme in complex with D-ornithine and in complex with the inhibitor 2,4-diaminobutyrate are determined; the structure of substrate-free ornithine 4,5-aminomutase is solved to a resolution of 2.0 A, furthermore the structures of the enzyme in complex with D-ornithine and in complex with the inhibitor 2,4-diaminobutyrate are determined
E3PY95, E3PY96
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
45
-
rapid inactivation above 45C
2074
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
glycerol stabilizes during storage and purification
-
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-20C, at a concentration of 0.7 mg/ml, 35% loss of activity after 1 month
-
-80C, the purified enzyme can be stored in concentrated solution in the presence of 50% glycerol for several months
-
4C, 20% glycerol v/v or 0.02 mM coenzyme B12, enzyme concentration 1.0 mg/ml, 9% loss of activity after 2 d
-
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
enzyme S subunit OraS, expression in Escherichia coli
-
Ni2+ -NTA column chromatography and Q-Sepharose resin chromatography
-
recombinant refolded D-ornithine aminomutase
-
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
enzyme S subunit OraS, expression in Escherichia coli
-
expressed in Escherichia coli strain Rosetta(DE3)pLysS
-
expression of oraA and oraE genes encoding D-ornithine aminomutase in Escherichia coli
-
for expression in Escherichia coli cells; for expression in Escherichia coli cells
E3PY95, E3PY96
genes oraE and oraS for subunits OraS and OraE, expression in Escherichia coli
-
recombinant expression of wild-type and mutant enzymes; recombinant expression of wild-type and mutant enzymes
-
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
E338A
-
site-directed mutagenesis, substrate binding of the mutant is unaffected, but kcat is reduced 670fold and catalytic efficiency 220fold compared to the wild-type enzyme. The rate of external aldimine formation in the mutant is similar to that of the wild-type enzyme, but it shows no detectable adenosylcobalamin homolysis upon binding of the physiological substrate
E338D
-
site-directed mutagenesis, substrate binding of the mutant is unaffected, but kcat is reduced 380fold and catalytic efficiency 60fold compared to the wild-type enzyme. The rate of external aldimine formation in the mutant is similar to that of the wild-type enzyme, but it shows n detectable adenosylcobalamin homolysis upon binding of the physiological substrate, site-directed mutagenesis, substrate binding of the mutant is unaffected, but kcat is reduced 380fold and catalytic efficiency 60fold compared to the wild-type enzyme. The rate of external aldimine formation in the mutant is similar to that of the wild-type enzyme, but it shows no detectable adenosylcobalamin homolysis upon binding of the physiological substrate
E338Q
-
site-directed mutagenesis, substrate binding of the mutant is unaffected, but kcat is reduced 90fold and catalytic efficiency 20fold compared to the wild-type enzyme. The rate of external aldimine formation in the mutant is similar to that of the wild-type enzyme, but it shows no detectable adenosylcobalamin homolysis upon binding of the physiological substrate
H225A
-
site-directed mutagenesis
H225A
Clostridium sticklandii DSM S19
-
site-directed mutagenesis
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H225Q
Clostridium sticklandii DSM S19
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site-directed mutagenesis
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H225Q
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site-directed mutagenesis
additional information
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protein KamDE comprised of the 30000 and 51000 Da subunits of the E1 component of D-alpha-lysine aminomutase is catalytically active in absence of the third 12800 kDa subunit, but ATP no longer has a regulatory effect on it. The S subunit of D-ornithine aminomutase, OraS, is capable of forming a complex with KamDE and restores the enzymes ATP-dependent allosteric regulation. OraS protein alone lowers the Km of KamDE for adenosylcobalamin and pyridoxal phosphate