Information on EC 5.4.99.1 - Methylaspartate mutase

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The expected taxonomic range for this enzyme is: Bacteria, Eukaryota

EC NUMBER
COMMENTARY
5.4.99.1
-
RECOMMENDED NAME
GeneOntology No.
Methylaspartate mutase
REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
L-threo-3-methylaspartate = L-glutamate
show the reaction diagram
-
-
-
-
L-threo-3-methylaspartate = L-glutamate
show the reaction diagram
the 4-glutamyl radical is an intermediate in the carbon skeleton rearrangement catalyzed the enzyme; the reaction is initiated through hydrogen atom abstraction from C-4 of Glu by 5'-deoxyadenosyl radical which is derived by homolysis of the Co-C sigma-bond of coenzyme B12
-
L-threo-3-methylaspartate = L-glutamate
show the reaction diagram
hydrogen transfer occurs directly between coenzyme and product and provides no evidence for the formation of a protein radical
-
L-threo-3-methylaspartate = L-glutamate
show the reaction diagram
mechanism
-
L-threo-3-methylaspartate = L-glutamate
show the reaction diagram
requires Co(II) for coordiation of the acrylate, in order to enable the addition of the radical fragment to the alpha-carbon of this intermediate, leading to the branched product
-
L-threo-3-methylaspartate = L-glutamate
show the reaction diagram
the catalytic mechanism proceeds via a fragmentation/recombination sequence with intermediates stabilized by partial protonation/deprotonation
-
L-threo-3-methylaspartate = L-glutamate
show the reaction diagram
kinetic study of tritium effects, reaction has a late transition state
-
L-threo-3-methylaspartate = L-glutamate
show the reaction diagram
study of Co-C bond activation, cofactor/active site interactions give rise to a fairly uniform stabilization of the Co 3d orbitals
-
L-threo-3-methylaspartate = L-glutamate
show the reaction diagram
kinetic study of deuterium effects, an isotopically insensitive step is partially rate-determining
-
L-threo-3-methylaspartate = L-glutamate
show the reaction diagram
no significant activation of coenzyme adenosylcobalamin in absence of substrate
-
L-threo-3-methylaspartate = L-glutamate
show the reaction diagram
motions of the 5-hydrogen atoms are coupled in the transition state to the motion of the hydrogen undergoing transfer, in a reaction that involves a large degree of quantum tunneling
-
REACTION TYPE
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
group transfer
-
-
intramolecular
-
isomerization
-
-
-
-
PATHWAY
KEGG Link
MetaCyc Link
C5-Branched dibasic acid metabolism
-
glutamate degradation VI (to pyruvate)
-
isoleucine biosynthesis III
-
Metabolic pathways
-
methylaspartate cycle
-
SYSTEMATIC NAME
IUBMB Comments
L-threo-3-Methylaspartate carboxy-aminomethylmutase
Requires a cobamide coenzyme.
SYNONYMS
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
AdoCbl-dependent glutamate mutase
-
-
Glm
P80077, P80078
-
Glutamate isomerase
-
-
-
-
Glutamate mutase
-
-
-
-
Glutamate mutase
-
-
Glutamate mutase
P80077, P80078
-
Glutamate mutase
-
-
Glutamic acid isomerase
-
-
-
-
Glutamic acid mutase
-
-
-
-
Glutamic isomerase
-
-
-
-
Glutamic mutase
-
-
-
-
Methylaspartic acid mutase
-
-
-
-
Mutase, methylaspartate
-
-
-
-
CAS REGISTRY NUMBER
COMMENTARY
9032-97-7
-
ORGANISM
COMMENTARY
LITERATURE
SEQUENCE CODE
SEQUENCE DB
SOURCE
; recombinant enzyme expressed in Escherichia coli
-
-
Manually annotated by BRENDA team
expression in Escherichia coli as fusion protein
-
-
Manually annotated by BRENDA team
methylaspartate mutase E chain (subunit epsilon)
-
Manually annotated by BRENDA team
methylaspartate mutase S chain (subunit sigma)
UniProt
Manually annotated by BRENDA team
recombinant enzyme expressed in Escherichia coli
-
-
Manually annotated by BRENDA team
Clostridium saccharobutyricum
-
-
-
Manually annotated by BRENDA team
Clostridium sp. SB4
SB4
-
-
Manually annotated by BRENDA team
no activity in Acidaminococcus fermentans
-
-
-
Manually annotated by BRENDA team
no activity in Clostridium microsporium
-
-
-
Manually annotated by BRENDA team
no activity in Fusobacterium fusiforme
-
-
-
Manually annotated by BRENDA team
no activity in Fusobacterium nucleatum
-
-
-
Manually annotated by BRENDA team
no activity in Micrococcus aerogenes
-
-
-
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
physiological function
-
overexpression of glmS leads to a 4.9fold enhancement of intracellular vitamin B12 concentration
SUBSTRATE
PRODUCT                      
REACTION DIAGRAM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
(Substrate)
LITERATURE
(Substrate)
COMMENTARY
(Product)
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
(S)-2-hydroxyglutarate
(2S,3S)-3-methylmalate
show the reaction diagram
-
-
analysis of the energy profile for the various intermediate steps of reaction
-
?
Glu
?
show the reaction diagram
-
the enzyme is involved in the dissimilation of Glu to acetyl-CoA and pyruvate through the following pathway
-
-
-
Glu
?
show the reaction diagram
-
initial reaction in anaerobic degradation of Glu
-
-
-
Glu
?
show the reaction diagram
-
first step in Glu fermentation pathway
-
-
-
Glu
?
show the reaction diagram
Clostridium sp. SB4
-
energy-yielding fermentation of Glu
-
-
-
L-2-hydroxyglutarate
L-threo-3-methylmalate
show the reaction diagram
-
rate-limiting step is most likely the rearrangement of the 2-hydroxyglutaryl radical to the 3-methylmalyl radical
-
?
L-Glu
threo-3-Methylaspartate
show the reaction diagram
-
-
-
-
L-Glu
threo-3-Methylaspartate
show the reaction diagram
-
-
-
-
L-Glu
threo-3-Methylaspartate
show the reaction diagram
-
-
-
-
L-Glu
threo-3-Methylaspartate
show the reaction diagram
-
-
-
-
L-Glu
threo-3-Methylaspartate
show the reaction diagram
-
-
-
-
L-Glu
threo-3-Methylaspartate
show the reaction diagram
-
-
-
-
L-Glu
threo-3-Methylaspartate
show the reaction diagram
-
-
-
-
L-Glu
threo-3-Methylaspartate
show the reaction diagram
-
-
-
-
L-Glu
threo-3-Methylaspartate
show the reaction diagram
-
-
-
-
L-Glu
threo-3-Methylaspartate
show the reaction diagram
-
-
-
-
L-Glu
threo-3-Methylaspartate
show the reaction diagram
-
-
-
-
L-Glu
threo-3-Methylaspartate
show the reaction diagram
-
-
-
-
L-Glu
threo-3-Methylaspartate
show the reaction diagram
-
-
-
-
L-Glu
threo-3-Methylaspartate
show the reaction diagram
-
-
-
-
L-Glu
threo-3-Methylaspartate
show the reaction diagram
-
-
-
-
L-Glu
threo-3-Methylaspartate
show the reaction diagram
-
-
-
-
L-Glu
threo-3-Methylaspartate
show the reaction diagram
-
-
-
-
L-Glu
threo-3-Methylaspartate
show the reaction diagram
-
-
-
-
L-Glu
threo-3-Methylaspartate
show the reaction diagram
-
-
-
-
L-Glu
threo-3-Methylaspartate
show the reaction diagram
-
-
-
-
L-Glu
threo-3-Methylaspartate
show the reaction diagram
-
-
-
-
L-Glu
threo-3-Methylaspartate
show the reaction diagram
-
-
-
-
L-Glu
threo-3-Methylaspartate
show the reaction diagram
Clostridium saccharobutyricum, Clostridium tetani
-
-
-
-
L-Glu
threo-3-Methylaspartate
show the reaction diagram
-
-
(2S,3S)-3-methylaspartate
-
L-Glu
threo-3-Methylaspartate
show the reaction diagram
-
-
(2S,3S)-3-methylaspartate
-
L-Glu
threo-3-Methylaspartate
show the reaction diagram
-
equilibrium favours glutamate formation
-
-
L-Glu
threo-3-Methylaspartate
show the reaction diagram
-
in the reverse reaction: erythro-3-methylaspartate does not serve as substrate, L-
-
-
L-Glu
threo-3-Methylaspartate
show the reaction diagram
-
high-level quantum chemistry calculations on catalytic reaction. Overall reaction is exothermic by 5.2 kJ per mol, while the equilibrium for the reaction in aqueous solution lies in the opposite direction
-
-
?
L-Glu
threo-3-Methylaspartate
show the reaction diagram
Clostridium sp. SB4
-
-
-
-
L-Glu
L-threo-3-methylaspartate
show the reaction diagram
-
-
-
-
?
L-Glu
L-threo-3-methylaspartate
show the reaction diagram
P80077, P80078
-
-
-
r
L-glutamate
L-threo-3-methylaspartate
show the reaction diagram
-
-
-
-
?
L-glutamate
L-threo-3-methylaspartate
show the reaction diagram
-
-
-
?
L-glutamate
L-threo-3-methylaspartate
show the reaction diagram
-
-
-
?
L-glutamate
L-threo-3-methylaspartate
show the reaction diagram
-
-
-
?
L-glutamate
L-threo-3-methylaspartate
show the reaction diagram
-
-
-
-
?
L-glutamate
L-threo-3-methylaspartate
show the reaction diagram
-
kinetic competence of acrylate and glycyl radical as intermediates in the rearrangement of glutamate to methylaspartate
-
?
L-glutamate
threo-3-methyl-L-aspartate
show the reaction diagram
-
-
-
-
?
L-threo-3-methylaspartate
L-glutamate
show the reaction diagram
-
-
-
-
r
additional information
?
-
-
the catalytic mechanism proceeds via a fragmentation/recombination sequence with intermediates stabilized by partial protonation/deprotonation
-
?
NATURAL SUBSTRATES
NATURAL PRODUCTS
REACTION DIAGRAM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
(Substrate)
LITERATURE
(Substrate)
COMMENTARY
(Product)
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
Glu
?
show the reaction diagram
-
the enzyme is involved in the dissimilation of Glu to acetyl-CoA and pyruvate through the following pathway
-
-
-
Glu
?
show the reaction diagram
-
initial reaction in anaerobic degradation of Glu
-
-
-
Glu
?
show the reaction diagram
-
first step in Glu fermentation pathway
-
-
-
Glu
?
show the reaction diagram
Clostridium sp. SB4
-
energy-yielding fermentation of Glu
-
-
-
COFACTOR
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
adenosylcobalamin
-
Km-value for wild-type enzyme is 0.005 mM. No cob(II)alamin detected in UV-visible spectrum of mutant enzymes R100M and R100Y. In mutant enzyme R100K cob(II)alamin accumulates to a concentration similar to that of the wild-type enzyme, homolysis of the coenzyme is slower by an order of magnitude, compared to wild-type enzyme. Mutant does not exhibit the very large deuterium isotope effects that are observed for homolysis of the coenzyme when the wild-type enzyme is reacted with deuterated substrates
adenosylcobalamin
-
enzyme is dependent on, KM for wild-type enzyme is 0.0055 mM
adenosylcobalamin
-
study of tritium isotope effects on homolysis of adenosylcobalamin
adenosylcobalamin
-
study of Co-C bond activation, cofactor/active site interactions give rise to a fairly uniform stabilization of the Co 3d orbitals
adenosylcobalamin
-
kinetics of homolysis and recombination, ultrafast spectroscopic experiments, no significant activation of coenzyme in absence of substrate
adenosylcobalamin
-
investigation of photolysis by transient absorption spectroscopy. Metal-to-ligand charge transfer intermediate decays to form cob(II)alamin with a time constand of 105 ps
adenosylcobalamin
-
dependent on
adenosylcobalamin
-
dependent on
adenosylcobalamin
P80077, P80078
;
adenosylcobalamin
-
-
Co-Deoxycobalamin
-
essential for the reaction
Cobalamin
-
dependent on
Cobalamin
-
and at least seven analogs containing bases other than dimethylbenzimidazolyl can serve as coenzymes
Cobalamin
-
the weakly associated subunits E and S combine to form the coenzyme binding site. Interactions between the protein and the adenosyl moiety do not serve to weaken the cobalt-carbon bond in the ground state, variation of the apparent Km-value for adenosylcobalamine with protein concentration. Km: 0.0055 mM in the reaction with L-Glu, Km: 0.0031 mM in the reaction with (2S,3S)-3-methylaspartate, genetically engineered enzyme with S subunit fused to the C-terminus of the E subunit through an 11 amino acid (Gly-Gln)5-Gly linker segment
Cobalamin
-
dependent on; requires Co(II) for coordination of the acrylate, in order to enable the addition of the radical fragment to the alpha-carbon of this intermediate, leading to the branched product
Cobalamin
-
coenzyme binding and catalysis is very sensitive to mutations at position 15; dependent on
Cobalamin
-
component S binds coenzyme B12; dependent on
Cobalamin
-
dependent on; the major part of component S is preorganized for vitamin B12 binding, but the B12-binding site itself is only partially formed. Upon binding B12, important elements of the binding site appear to become structured, including an alpha-helix that forms one side of the cleft accomodating the nucleotide 'tail' of the cofactor
coenzyme B12
-
the alpha2beta2 tetramer consists of two subunits, subunit MutS of 53600 Da and subunit GlmS of 14800 Da, whose assembly is mediated by coenzyme B12. In GlnS and MutS the sequence motif, Asp-Xaa-His-Xaa-Xaa-Gly, which includes the cobalt-coordinating histidine residue, and a predicted alpha-helical region following the motif, are present as an unstructured and highly mobile loop. In the absence of coenzyme, the B12-binding site apparently is only partially formed. Important elements of the binding site only become structured upon binding B12, these include the cobalt-coordinating histidine residue, and an alpha helix that forms one side of the cleft accomodating the nucleotide tail of the coenzyme
coenzyme B12
-
the coenzyme B12-binding subunit traps the nucleotide moiety of coenzyme B12
coenzyme B12
-
each of the two independent coenzyme B12 molecules is associated with a substrate-binding site
coenzyme B12
-
binds to subunit SanU with about 0.1 mol Co per mol SanU
METALS and IONS
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
Co2+
-
0.1 mol per mol of subunit SanU
INHIBITORS
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
(2S)-Homocysteic acid
-
-
(2S,3R)-3-Fluoroglutamate
-
-
(2S,3R)-3-Methylglutamate
-
-
(2S,3S)-3-Methylglutamate
-
-
(2S,4S)-4-Fluoroglutamate
-
-
(2S,4S)-4-Fluoroglutamic acid
-
-
(2S,4S)-4-Fluoroglutamic acid
-
-
(S)-2-thiolglutaric acid
-
high-level quantum chemical calculations of reaction intermediates
(S)-3-Methylitaconate
-
-
(S)-3-Methylitaconic acid
-
-
1-Bromo-cyclopropane-cis-1,2-diacidic acid
-
-
1-Bromo-cyclopropane-trans-1,2-diacidic acid
-
-
2-Bromo-2,3-Methanosuccinic acid
-
-
2-ketoglutaric acid
-
high-level quantum chemical calculations of reaction intermediates
2-Methyleneglutarate
-
binding of the 2-methyleneglutarate to glutamate mutase initiates homolysis of adensosylcobalamin, irreversible inhibition
2-Methyleneglutaric acid
-
-
2-Methyleneglutaric acid
-
-
2-oxoglutarate
-
inactivates, formation of the C-4 radical of 2-oxoglutarate is a facile process, but it does not undergo further reactions
2-thiolglutarate
-
competitive. 2-Thiolglutarate elicits cobalt-carbon bond homolysis and the formation of 5-deoxyadenosine. 2-Thiolglutarate first forms a thiolglutaryl radical at C-4 that then undergoes fragmentation to produce acrylate and the sulfur-stabilized thioglycolyl radical which accumulates on the enzyme
Hog intrinsic factor
-
coenzyme B12 restores activity
-
iodoacetate
-
component S is inhibited, component E is not inhibited
iodoacetate
-
specifically alkylates Cys15 in enzyme component S with concomitant irreversible loss of enzyme activity
ACTIVATING COMPOUND
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
2-mercaptoethanol
-
component S requires treatment with 2-mercaptoethanol to show maximal activity. Component E does not require this treatment
Benzimidazolribofuranosyl-adenosylcobinamide
-
can serve as cofactor, Km-value in reaction with L-Glu, fusion protein in which the cobalamin-binding subunit is linked to the catalytic subunit: 0.0048 mM. Km-value in reaction with L-Glu, wild-type enzyme: 0.0005 mM
additional information
-
overview: activity with modified coenzymes
-
KM VALUE [mM]
KM VALUE [mM] Maximum
SUBSTRATE
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
0.14
-
3-methylaspartate
-
genetically engineered enzyme with S subunit fused to the C-terminus of the E-subunit through an 11 amino acid (Gly-Gln)5-Gly linker segment
0.5
-
3-methylaspartate
-
-
0.5
-
3-methylaspartate
-
-
0.5
-
3-methylaspartate
-
-
1.2
-
L-2-Hydroxyglutarate
-
pH 7.0
0.58
-
L-Glu
-
genetically engineered enzyme with S subunit fused to the C-terminus of the E-subunit through an 11 amino acid (Gly-Gln)5-Gly linker segment
1.2
-
L-Glu
-
reaction with benzimidazolribofuranosyl-adenosylcobinamide, wild-type enzyme and fusion protein in which the cobalamin-binding subunit is linked to the catalytic subunit
0.24
-
L-glutamate
-
23C, mutant enzyme E171Q
0.38
-
L-glutamate
-
23C, mutant enzyme E171D
0.58
-
L-glutamate
-
25C, wild-type enzyme
0.58
-
L-glutamate
-
23C, wild-type enzyme
0.65
-
L-glutamate
-
23C, mutant enzyme E171A
1.07
-
L-glutamate
-
23C, mutant enzyme E171N
7.6
-
L-glutamate
-
25C, mutant enzyme R100Y
10
-
L-glutamate
-
25C, mutant enzyme R100K
13
-
L-glutamate
-
25C, mutant enzyme R100M
0.14
-
L-threo-3-methylaspartate
-
wild-type, pH 7.0
5.2
-
L-threo-3-methylaspartate
-
37C
9.5
-
L-threo-3-methylaspartate
-
mutant R100K, pH 7.0
additional information
-
additional information
-
Km values for modified coenzymes
-
TURNOVER NUMBER [1/s]
TURNOVER NUMBER MAXIMUM[1/s]
SUBSTRATE
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
5.8
-
(2S,3S)-3-methylaspartate
-
genetically engineered enzyme with S subunit fused to the C-terminus of the E subunit through an 11 amino acid (Gly-Gln)5-Gly linker segment
0.05
-
L-2-Hydroxyglutarate
-
pH 7.0
0.65
-
L-Glu
-
reaction of L-Glu with benzimidazolribofuranosyl-adenosylcobinamide, fusion protein in which the cobalamin-binding subunit is linked to the catalytic subunit
5.8
-
L-Glu
-
genetically engineered enzyme with S subunit fused to the C-terminus of the E subunit through an 11 amino acid (Gly-Gln)5-Gly linker segment
20
-
L-Glu
-
reaction of L-Glu with benzimidazolribofuranosyl-adenosylcobinamide, wild-type enzyme
0.0181
-
L-glutamate
-
25C, mutant enzyme R100Y
0.0211
-
L-glutamate
-
25C, mutant enzyme R100M
0.025
-
L-glutamate
-
23C, mutant enzyme E171N
0.048
-
L-glutamate
-
25C, mutant enzyme R100K
0.11
-
L-glutamate
-
23C, mutant enzyme E171Q
0.21
-
L-glutamate
-
23C, mutant enzyme E171A
3
6
L-glutamate
-
23C, mutant enzyme E171D
3.19
-
L-glutamate
-
23C, mutant enzyme E171D
5.8
-
L-glutamate
-
25C, wild-type enzyme
5.8
-
L-glutamate
-
23C, wild-type enzyme
0.073
-
L-threo-3-methylaspartate
-
mutant R100K, pH 7.0
5.4
-
L-threo-3-methylaspartate
-
wild-type, pH 7.0
Ki VALUE [mM]
Ki VALUE [mM] Maximum
INHIBITOR
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
0.05
-
2-thiolglutarate
-
pH 7.0
SPECIFIC ACTIVITY [µmol/min/mg]
SPECIFIC ACTIVITY MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
additional information
-
-
component E
pH OPTIMUM
pH MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
7
-
-
protein with the S subunit genetically fused to the C-terminus of the E-subunit through an 11 amino acid (Gly-Gln)5-Gly linker segment
8
8.5
-
wild type enzyme
pH RANGE
pH RANGE MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
6.5
8.7
-
pH 6.5: about 50% of maximal activity, pH 8.7: about 80% of maximal activity
7.5
9
-
activity drops sharply at pH 7.5 and pH 9.0
TEMPERATURE OPTIMUM
TEMPERATURE OPTIMUM MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
40
-
-
maximal activity in a 30 min incubation period
55
-
-
maximal activity in an incubation period that is shorter than 30 min
TEMPERATURE RANGE
TEMPERATURE MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
25
37
-
the reaction rate increases about 3fold when the temperature is increased from 25C to 37C
27
38
-
27C: about 50% of maximal activity, 38C: maximal activity
SUBUNITS
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
?
-
the enzyme consists of two separable proteins called component E and component S, MW of component E determined by ultracentrifugation is 128000, the molecular weight of component S determined by ultracentrifugation is 17000
?
-
enzyme is composed of two components: E, a dimer, epsilon2, and S, a monomer, sigma = 14700
?
-
x * component E = MW 50000 + x * component S = 6000, SDS-PAGE
?
-
component E is a dimer, epsilon2, with epsilon = MW 53500, + component S is a monomer, sigma = 14800
?
-
x * 18000, SDS-PAGE, x * 18370, calcultated, subunit SanU, x * 48000, SDS-PAGE, x * 47850, calculated, subunit SanV
tetramer
-
2 * 14800, B12-binding component S, + 2 * 53700, component E
heterodimer
-
1 * 53700 + 1 * 14000, two weakly-associating subunits, MutS and MutE, combine with adenosylcobalamin to form the active holoenzyme
additional information
-
no enzymic activity for subunit SanU or SanV alone, enzymic activity results after mixing both subunits plus coenzyme B12, reversible and dynamical equilibrium between SanU and SanV in formation of a possibly tetrameric complex
POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
additional information
-
sulfhydryl compounds are not essential for maintaining active SanU, but for activity of reassembled enzyme compolex
Crystallization/COMMENTARY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
crystal structure of inactive recombinant enzyme reconstituted with either cyanocobalamin or methylcobalamin, hanging-drop method
-
crystallization of component S, hanging drop method, polyethylene glycol 4000 as precipitant; recombinant enzyme
-
TEMPERATURE STABILITY
TEMPERATURE STABILITY MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
55
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rapidly inactivated
STORAGE STABILITY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
-20C, component E in presence of 50% glycerol, stable for several months
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4C or -20C, component S in concentrated solution in the absence of thiols, stable for several months
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Purification/COMMENTARY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
purification of component E and component S
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cloning and overexpression in Escherichia coli allows component E to be obtained in homogeneous form, free of inhibiting cobamides and traces of component S
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fusion protein in which the cobalamin-binding subunit is linked to the catalytic subunit
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purification of component E and component S
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after individual expression of SanU and SanV in Escherichia coli
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Cloned/COMMENTARY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
expression in Escherichia coli
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overexpression of polypeptide chains sigma and epsilon in Escherichia coli
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polypeptide chains sigma and epsilon expressed in Escherichia coli
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13C,15N-labeled MutS, the coenzyme B12-binding subunit of glutamate mutase prepared by overexpression from Escherichia coli
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glutamate mutase S component MutS. Induction strategy to enhance the level of protein expression in bacteriophage T7 expression system in Escherichia coli. Yield of purified protein is increased threefold by reducing the induction temperature to 20C and using 0.2% lactose and 50 mg per l IPTG simultaneously as inducers
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overexpression of component E and S in Escherichia coli
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polypeptide chains sigma and epsilon expressed in Escherichia coli
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expressed in Escherichia coli
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individual expression of SanU and SanV in Escherichia coli
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ENGINEERING
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
E171A
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turnover number for glutamate is reduced 27.6fold, KM-value is increased 1.1fold, Km-value for adenosylcobalamin is reduced 1.23fold
E171D
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turnover number for glutamate is reduced 1.8fold, KM-value is reduced 1.54fold, Km-value for adenosylcobalamin is reduced 2.7fold
E171N
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turnover number for glutamate is reduced 232fold, KM-value is increased by 1.8fold, Km-value for adenosylcobalamin is reduced 1.4fold
R100K
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cob(II)alamin accumulates to a concentration similar to that of the wild-type enzyme, homolysis of the coenzyme is slower by an order of magnitude, compared to wild-type enzyme, glutamate binding is significantly weakened. Mutant does not exhibit the very large deuterium isotope effects that are observed for homolysis of the coenzyme when the wild-type enzyme is reacted with deuterated substrates. Km-value for glutamate is reduced 121fold compared to wild-type enzyme, KM-value for glutamate is increased 17fold compared to wild-type enzyme
R100K
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mutation significantly impairs the ability of enzyme to catalyze the rearrangement of substrate radical to product radical
R100M
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no cob(II)alamin detected in UV-visible spectrum. Km-value for glutamate is reduced 276fold compared to wild-type enzyme, KM-value for glutamate is increased 13fold compared to wild-type enzyme
R100Y
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no cob(II)alamin detected in UV-visible spectrum. Km-value for glutamate is reduced 322fold compared to wild-type enzyme, KM-value for glutamate is increased 17fold compared to wild-type enzyme
C15A
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Cys15Ser and Cys15Ala of enzyme component S are active, but exhibit decreased maximal velocity and increased apparent Km-value for adenosylcobalamin. Mutants Cys15Asp and Cys15Asn of component S of the methylaspartate are inactive
C15N
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Cys15Ser and Cys15Ala of enzyme component S are active, but exhibit decreased maximal velocity and increased apparent Km-value for adenosylcobalamin. Mutants Cys15Asp and Cys15Asn of component S of the methylaspartate are inactive
E171Q
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turnover number for glutamate is reduced 53fold, KM-value is reduced 2.4fold, Km-value for adenosylcobalamin is reduced 2fold, mutant enzyme is independent of pH
additional information
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the S subunit is genetically fused to the C-terminus of the E subunit through an 11 amino acid 5-Gly linker segment. The affinity of adenosylcobalamine is unchanged, but the turnover-number and the Km-value for Glu in the conversion of L-Glu to (2S,3S)-3-methylaspartate are decreased by about a third
C15S
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Cys15Ser and Cys15Ala of enzyme component S are active, but exhibit decreased maximal velocity and increased apparent Km-value for adenosylcobalamin. Mutants Cys15Asp and Cys15Asn of component S of the methylaspartate are inactive
additional information
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fusion protein in which the cobalamin-binding subunit is linked to the catalytic subunit
APPLICATION
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
synthesis
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induction strategy to enhance the level of protein expression in bacteriophage T7 expression system in Escherichia coli. Yield of purified glutamate mutase S component MutS protein is increased threefold by reducing the induction temperature to 20C and using 0.2% lactose and 50 mg per l IPTG simultaneously as inducers