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 hide
5.4.99.1
-
RECOMMENDED NAME
GeneOntology No.
Methylaspartate mutase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
L-threo-3-methylaspartate = L-glutamate
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
group transfer
-
-
intramolecular
-
isomerization
-
-
-
-
PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
C5-Branched dibasic acid metabolism
-
-
glutamate and glutamine metabolism
-
-
Glyoxylate and dicarboxylate metabolism
-
-
isoleucine metabolism
-
-
L-glutamate degradation VI (to pyruvate)
-
-
L-isoleucine biosynthesis III
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-
Metabolic pathways
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methylaspartate cycle
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-
SYSTEMATIC NAME
IUBMB Comments
L-threo-3-Methylaspartate carboxy-aminomethylmutase
Requires a cobamide coenzyme.
CAS REGISTRY NUMBER
COMMENTARY hide
9032-97-7
-
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
Clostridium saccharobutyricum
-
-
-
Manually annotated by BRENDA team
SB4
-
-
Manually annotated by BRENDA team
SB4
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
-
-
-
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
COMMENTARY hide
LITERATURE
metabolism
physiological function
-
overexpression of glmS leads to a 4.9fold enhancement of intracellular vitamin B12 concentration
additional information
-
residue Glu117 and the arginine claw have a strong influence, and also residues Glu 214, Lys 322, Gln 147, Glu 330, Lys 326, and Met 294 play a catalytic role. The arginine claw keeps the intermediates in place and is probably responsible for the enantioselectivity. Glu 171 temporarily accepts a proton from the glutamyl radical intermediate and donates it back at the end of the reaction
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
(2S,3S)-3-methylaspartate
L-glutamate
show the reaction diagram
-
-
-
-
r
(S)-2-hydroxyglutarate
(2S,3S)-3-methylmalate
show the reaction diagram
-
-
analysis of the energy profile for the various intermediate steps of reaction
-
?
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
L-threo-3-methylaspartate
show the reaction diagram
L-Glu
threo-3-Methylaspartate
show the reaction diagram
L-glutamate
L-threo-3-methylaspartate
show the reaction diagram
L-glutamate
threo-3-methyl-L-aspartate
show the reaction diagram
-
-
-
-
?
L-threo-3-methylaspartate
L-glutamate
show the reaction diagram
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
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
(2S,3S)-3-methylaspartate
L-glutamate
show the reaction diagram
-
-
-
-
r
L-threo-3-methylaspartate
L-glutamate
show the reaction diagram
COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
5'-deoxyadenosylcobalamin
adenosylcobalamin
Co-Deoxycobalamin
-
essential for the reaction
Cobalamin
coenzyme B12
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
(2S)-Homocysteic acid
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-
(2S,3R)-3-Fluoroglutamate
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-
(2S,3R)-3-Methylglutamate
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-
(2S,3S)-3-Methylglutamate
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-
(2S,4S)-4-Fluoroglutamate
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-
(2S,4S)-4-Fluoroglutamic acid
(S)-2-thiolglutaric acid
-
high-level quantum chemical calculations of reaction intermediates
(S)-3-Methylitaconate
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-
(S)-3-Methylitaconic acid
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1-Bromo-cyclopropane-cis-1,2-diacidic acid
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1-Bromo-cyclopropane-trans-1,2-diacidic acid
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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-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
iodoacetate
Itaconic acid
additional information
-
charcoal adsorbs coenzyme B12 and inactivates glutamate mutase
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ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
2-mercaptoethanol
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component S requires treatment with 2-mercaptoethanol to show maximal activity. Component E does not require this treatment
Benzimidazolribofuranosyl-adenosylcobinamide
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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
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.14 - 0.5
3-methylaspartate
1.2
L-2-Hydroxyglutarate
-
pH 7.0
0.58 - 1.5
L-Glu
0.24 - 13
L-glutamate
0.14 - 9.5
L-threo-3-methylaspartate
additional information
additional information
-
Km values for modified coenzymes
-
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
5.8
(2S,3S)-3-methylaspartate
Clostridium cochlearium
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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
Clostridium cochlearium
-
pH 7.0
0.65 - 20
L-Glu
0.0181 - 6
L-glutamate
0.073 - 5.4
L-threo-3-methylaspartate
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.05
2-thiolglutarate
-
pH 7.0
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
-
component E
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
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
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6.5 - 8.7
-
pH 6.5: about 50% of maximal activity, pH 8.7: about 80% of maximal activity
7.5 - 9
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activity drops sharply at pH 7.5 and pH 9.0
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
40
-
maximal activity in a 30 min incubation period
55
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maximal activity in an incubation period that is shorter than 30 min
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
25 - 37
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the reaction rate increases about 3fold when the temperature is increased from 25C to 37C
27 - 38
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27C: about 50% of maximal activity, 38C: maximal activity
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
heterodimer
-
1 * 53700 + 1 * 14000, two weakly-associating subunits, MutS and MutE, combine with adenosylcobalamin to form the active holoenzyme
tetramer
-
2 * 14800, B12-binding component S, + 2 * 53700, component E
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
COMMENTARY hide
LITERATURE
additional information
-
sulfhydryl compounds are not essential for maintaining active SanU, but for activity of reassembled enzyme compolex
Crystallization/COMMENTARY
ORGANISM
UNIPROT
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
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
55
-
rapidly inactivated
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-20C, component E in presence of 50% glycerol, stable for several months
-
4C or -20C, component S in concentrated solution in the absence of thiols, stable for several months
-
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
after individual expression of SanU and SanV in Escherichia coli
-
cloning and overexpression in Escherichia coli allows component E to be obtained in homogeneous form, free of inhibiting cobamides and traces of component S
-
fusion protein in which the cobalamin-binding subunit is linked to the catalytic subunit
-
purification of component E and component S
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
13C,15N-labeled MutS, the coenzyme B12-binding subunit of glutamate mutase prepared by overexpression from Escherichia coli
-
expressed in Escherichia coli
-
expression in Escherichia coli
-
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
-
individual expression of SanU and SanV in Escherichia coli
-
overexpression of component E and S in Escherichia coli
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overexpression of polypeptide chains sigma and epsilon in Escherichia coli
-
polypeptide chains sigma and epsilon expressed in Escherichia coli
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
E171A
-
turnover number for glutamate is reduced 27.6fold, KM-value is increased 1.1fold, Km-value for adenosylcobalamin is reduced 1.23fold
E171D
-
turnover number for glutamate is reduced 1.8fold, KM-value is reduced 1.54fold, Km-value for adenosylcobalamin is reduced 2.7fold
E171N
-
turnover number for glutamate is reduced 232fold, KM-value is increased by 1.8fold, Km-value for adenosylcobalamin is reduced 1.4fold
E171Q
-
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
R100M
-
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
-
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
-
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
-
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
C15S
-
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
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
synthesis
-
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
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