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Information on EC 5.4.99.1 - methylaspartate mutase and Organism(s) Clostridium tetanomorphum and UniProt Accession Q05488
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5.4.99.1 methylaspartate mutaseIUBMB Comments
Requires a cobamide coenzyme.
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Word Map
- 5.4.99.1
- adenosylcobalamin
-
adenosylcobalamin-dependent
-
homolysis
-
b12-dependent
-
cobiialamin
- tetanomorphum
-
cobalt-carbon
- cochlearium
-
s-glutamate
-
b12-binding
-
2s,3s-3-methylaspartate
- 5'-deoxyadenosine
-
corrin
-
adenosyl
-
cobalamin-binding
-
cobalamin-dependent
-
methylmalonyl
-
isomerizations
- mutases
-
5\'-deoxyadenosyl
- 2-methyleneglutarate
- mesaconate
- dimethylbenzimidazole
- synthesis
The taxonomic range for the selected organisms is: Clostridium tetanomorphum
The expected taxonomic range for this enzyme is: Bacteria, Archaea, Eukaryota
The expected taxonomic range for this enzyme is: Bacteria, Archaea, Eukaryota
Synonyms
glutamate mutase, adocbl-dependent glutamate mutase, 
more
topSYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
Glutamate isomerase
-
-
-
-
Glutamate mutase
Glutamic acid isomerase
-
-
-
-
Glutamic acid mutase
-
-
-
-
Glutamic isomerase
-
-
-
-
Glutamic mutase
-
-
-
-
Methylaspartic acid mutase
-
-
-
-
Mutase, methylaspartate
-
-
-
-
Glutamate mutase
-
-
-
-
REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT
LITERATURE
L-threo-3-methylaspartate = L-glutamate
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
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
-
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
isomerization
-
-
-
-
PATHWAY SOURCE
PATHWAYS
KEGG
-
-, -, -
SYSTEMATIC NAME
IUBMB Comments
L-threo-3-Methylaspartate carboxy-aminomethylmutase
Requires a cobamide coenzyme.
CAS REGISTRY NUMBER
COMMENTARY
9032-97-7
-
SUBSTRATE
PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
L-Glu
threo-3-Methylaspartate
-
equilibrium favours glutamate formation
-
?
L-Glu
threo-3-Methylaspartate
-
in the reverse reaction: erythro-3-methylaspartate does not serve as substrate
-
?
NATURAL SUBSTRATE
NATURAL PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
COFACTOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
Cobalamin
-
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
Cobalamin
-
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
Cobalamin
-
and at least seven analogs containing bases other than dimethylbenzimidazolyl can serve as coenzymes
coenzyme B12
-
the coenzyme B12-binding subunit traps the nucleotide moiety of coenzyme B12
coenzyme B12
adenosylcobalamin, the concentration of internal coenzyme B12 is crucial for the full activity of B12-dependent enzyme
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
iodoacetate
-
specifically alkylates Cys15 in enzyme component S with concomitant irreversible loss of enzyme activity
ACTIVATING COMPOUND
ORGANISM
UNIPROT
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]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
additional information
additional information
-
Km values for modified coenzymes
-
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
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
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
20
L-Glu
-
reaction of L-Glu with benzimidazolribofuranosyl-adenosylcobinamide, wild-type enzyme
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
pH RANGE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
55
-
maximal activity in an incubation period that is shorter than 30 min
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
25 - 37
-
the reaction rate increases about 3fold when the temperature is increased from 25°C to 37°C
27 - 38
-
27°C: about 50% of maximal activity, 38°C: maximal activity
ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
-
3448, 3450, 3451, 3452, 3453, 3454, 3456, 3457, 3458, 3459, 3460, 3464, 650853, 652857, 679538, 692318
-
-
small sigma subunit and large epsilon subunit, respectively
UniProt
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
additional information
additional information
identification of the reactivating factor of glutamate mutase, MutL. MutL regenerates glutamate mutase and releases inactive coenzyme from the inactive glutamate mutase-adenosylcobalamine complex. Reactivation of the spontaneous inactive mutase (mutase-X-Cbl) and inactive form B12 binding with mutase (mutase-CNCbl) in presence of ATP and coenzyme B12 by MutL
additional information
-
identification of the reactivating factor of glutamate mutase, MutL. MutL regenerates glutamate mutase and releases inactive coenzyme from the inactive glutamate mutase-adenosylcobalamine complex. Reactivation of the spontaneous inactive mutase (mutase-X-Cbl) and inactive form B12 binding with mutase (mutase-CNCbl) in presence of ATP and coenzyme B12 by MutL
UNIPROT
ENTRY NAME
ORGANISM
NO. OF AA
NO. OF TRANSM. HELICES
MOLECULAR WEIGHT[Da]
SOURCE
SEQUENCE
LOCALIZATION PREDICTION
The reliability class of the localization prediction ranges from 1 (very reliable) to 5 (not reliable).
The reliability class of the localization prediction ranges from 1 (very reliable) to 5 (not reliable). PDB
SCOP
CATH
UNIPROT
ORGANISM
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
14000
-
1 * 53700 + 1 * 14000, two weakly-associating subunits, MutS and MutE, combine with adenosylcobalamin to form the active holoenzyme
53700
53700
-
1 * 53700 + 1 * 14000, two weakly-associating subunits, MutS and MutE, combine with adenosylcobalamin to form the active holoenzyme
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
heterodimer
tetramer
-
2 * 14800, B12-binding component S, + 2 * 53700, component E
?
-
enzyme is composed of two components: E, a dimer, epsilon2, and S, a monomer, sigma = 14700
?
-
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
heterodimer
-
1 * 53700 + 1 * 14000, two weakly-associating subunits, MutS and MutE, combine with adenosylcobalamin to form the active holoenzyme
heterodimer
the enzyme contains a small sigma subunit and a large epsilon subunit
CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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
additional information
-
fusion protein in which the cobalamin-binding subunit is linked to the catalytic subunit
additional information
production of mesaconate in Escherichia coli by engineered glutamate mutase pathway, establishment of mesaconate pathway in Escherichia coli. First, glutamate is synthesized from glucose via glycolysis and TCA cycle.Then glutamate is converted into 3-methylaspartate by glutamate mutase. Finally, mesaconate is formed by elimination of ammonia from 3-methylaspartate via MAL. Since Escherichia coli does not contain glutamate mutase and 3-methylaspartate ammonia lyase, the two enzymes from Clostridium tetanomorphum are heterologously expressed. To increase the flux from glutamate to mesaconate, two effective strategies are employed to optimize the critical enzyme activity in the pathway: one is regenerating inactive mutase. The other is enhancing the availability of glutamate mutase (stability) and coenzyme B12 (regeneration). For the highest mesaconate production strain EM9, the consumed glutamate is 6.91 g/l (40.9 mM) and mesaconate titer is 7.81 g/l (60 mM). The GlmE from Clostridium cochlearium shows best performance in mesaconate titer because GlmE is more stable than MutE
additional information
-
production of mesaconate in Escherichia coli by engineered glutamate mutase pathway, establishment of mesaconate pathway in Escherichia coli. First, glutamate is synthesized from glucose via glycolysis and TCA cycle.Then glutamate is converted into 3-methylaspartate by glutamate mutase. Finally, mesaconate is formed by elimination of ammonia from 3-methylaspartate via MAL. Since Escherichia coli does not contain glutamate mutase and 3-methylaspartate ammonia lyase, the two enzymes from Clostridium tetanomorphum are heterologously expressed. To increase the flux from glutamate to mesaconate, two effective strategies are employed to optimize the critical enzyme activity in the pathway: one is regenerating inactive mutase. The other is enhancing the availability of glutamate mutase (stability) and coenzyme B12 (regeneration). For the highest mesaconate production strain EM9, the consumed glutamate is 6.91 g/l (40.9 mM) and mesaconate titer is 7.81 g/l (60 mM). The GlmE from Clostridium cochlearium shows best performance in mesaconate titer because GlmE is more stable than MutE
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-20°C, component E in presence of 50% glycerol, stable for several months
-
4°C or -20°C, component S in concentrated solution in the absence of thiols, stable for several months
-
PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
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
-
recombinant His-tagged enzyme from Escherichia coli strain Rosetta2 (DE3) pLysS by nickel affinity chromatograpyh and ultrafiltration, recombinant expression in Escherichia coli strain BW25113, coexpression with Clostridium tetanomorphum 3-methylaspartate ammonia lyase
CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
13C,15N-labeled MutS, the coenzyme B12-binding subunit of glutamate mutase prepared by overexpression from Escherichia coli
-
genes mutS and mutE, recombinant expression of His-tagged enzyme in Escherichia coli strain Rosetta2 (DE3) pLysS
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 20°C and using 0.2% lactose and 50 mg per l IPTG simultaneously as inducers
-
APPLICATION
ORGANISM
UNIPROT
COMMENTARY
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 20°C and using 0.2% lactose and 50 mg per l IPTG simultaneously as inducers
REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Eagar, R.G.; Baltimore, B.G.; Herbst, M.M.; Barker, H.A.; Richards, J.H.
Mechanism of action of coenzyme B12. Hydrogen transfer in the isomerization of beta-methylaspartate to glutamate
Biochemistry
11
253-264
1972
Clostridium tetanomorphum
Switzer, R.L.
Glutamate mutase
B12 (Dolphin, D. ed. ) Wiley, New York
2
289-305
1982
Clostridium cochlearium, Clostridium saccharobutyricum, Clostridium sp., Acetoanaerobium sticklandii, Clostridium tetani, Clostridium tetanomorphum, no activity in Acidaminococcus fermentans, no activity in Clostridium microsporum, no activity in Fusobacterium fusiforme, no activity in Fusobacterium nucleatum, no activity in Micrococcus aerogenes, Cereibacter sphaeroides, Rhodospirillum rubrum, Clostridium sp. SB4
-
Barker, H.A.
beta-Methylaspartate-glutamate mutase from Clostridium tetanomorphum
Methods Enzymol.
113
121-133
1985
Clostridium tetanomorphum
Barker, H.A.
Coenzyme B12-dependent mutases causing carbon chain rearrangements
The Enzymes, 3rd Ed. (Boyer, P. D. , ed. )
6
509-537
1972
Cereibacter sphaeroides, Clostridium cochlearium, Clostridium tetanomorphum, Rhodospirillum rubrum
-
Marsh, E.N.G.
Tritium isotope effects in adenosylcobalamin-dependent glutamate mutase: implications for the mechanism
Biochemistry
34
7542-7547
1995
Clostridium tetanomorphum
Hartzoulakis, B.; Gani, D.
The mechanism of glutamate mutase: an unusually substrate-specific enzyme
Proc. Indian Acad. Sci. Chem. Sci.
106
1165-1176
1994
Clostridium tetanomorphum
-
Buckel, W.; Golding, B.T.
Glutamate and 2-methyleneglutarate mutase: from microbial curiosities to paradigms for coenzyme B12-dependent enzymes
Chem. Soc. Rev.
25
329-337
1996
Clostridium cochlearium, Clostridium tetanomorphum
-
Holloway, D.E.; Harding, S.E.; Marsh, E.N.G.
Adenosylcobalamin-dependent glutamate mutase: properties of a fusion protein in which the cobalamin-binding subunit is linked to the catalytic subunit
Biochem. J.
320
825-830
1996
Clostridium tetanomorphum
Holloway, D.E.; Chen, H.P.; Marsh, E.N.G.
Carboxymethylation of MutS-cysteine-15 specifically inactivates adenosylcobalamin-dependent glutamate mutase
J. Biol. Chem.
271
29121-29125
1996
Clostridium tetanomorphum
Holloway, D.E.; Marsh, E.N.G.
Adenosylcobalamin-dependent glutamate mutase from Clostridium tetanomorphum. Overexpression in Escherichia coli, purification, and characterization of the recombinant enzyme
J. Biol. Chem.
269
20425-20430
1994
Clostridium tetanomorphum
Tollinger, M.; Konrat, R.; Hilbert, B.H.; Marsh, E.N.; Krautler, B.
How a protein prepares for B12 binding: structure and dynamics of the B12-binding subunit of glutamate mutase from Clostridium tetanomorphum
Structure
15
1021-1033
1998
Clostridium tetanomorphum
Hoffmann, B.; Tollinger, M.; Konrat, R.; Huhta, M.; Marsh, E.N.G.; Krautler, B.
A protein pre-organized to trap the nucleotide moiety of coenzyme B12: refined solution structure of the B12-binding subunit of glutamate mutase from Clostridium tetanomorphum
ChemBioChem
2
643-655
2001
Clostridium tetanomorphum
Tollinger, M.; Eichmuller, C.; Konrat, R.; Huhta, M.S.; Marsh, E.N.; Krautler, B.
The B(12)-binding subunit of glutamate mutase from Clostridium tetanomorphum traps the nucleotide moiety of coenzyme B(12)
J. Mol. Biol.
309
777-791
2001
Clostridium tetanomorphum
Weng, Y.; Hsu, F.; Yang, W.; Chen, H.
Optimization of the overexpression of glutamate mutase S component under the control of T7 system by using lactose and IPTG as the inducers
Enzyme Microb. Technol.
38
465-469
2006
Clostridium tetanomorphum
-
Chen, H.P.; Hsu, H.J.; Hsu, F.C.; Lai, C.C.; Hsu, C.H.
Interactions between coenzyme B analogs and adenosylcobalamin-dependent glutamate mutase from Clostridium tetanomorphum
FEBS J.
275
5960-5968
2008
Clostridium tetanomorphum
Wang, J.; Zhang, K.
Production of mesaconate in Escherichia coli by engineered glutamate mutase pathway
Metab. Eng.
30
190-196
2015
no activity in Escherichia coli, Clostridium tetanomorphum (Q05488 AND Q05509), Clostridium tetanomorphum, Clostridium tetanomorphum ATCC 15920 (Q05488 AND Q05509)
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