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Information on EC 5.4.3.5 - D-ornithine 4,5-aminomutase
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5.4.3.5 D-ornithine 4,5-aminomutaseIUBMB Comments
A pyridoxal-phosphate protein that requires a cobamide coenzyme for activity.
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Word Map
- 5.4.3.5
- pyridoxal
- sticklandii
-
adocbl
-
homolysis
- 5'-phosphate
-
plp-dependent
-
5,6-aminomutase
-
aldimine
-
rossmann
-
radical-based
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stopped-flow
-
5\'-deoxyadenosyl
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paramagnetic
-
1,2-amino
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deprotonated
- d-lysine
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cobalamin-binding
-
rupture
- ribose
- imine
-
interconverting
-
5\'-deoxyadenosylcobalamin
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multistep
-
adocbl-dependent
-
isomerization
- pyridoxal-5'-phosphate
- pyridine
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uv-visible
-
refolding
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synergy
-
b12-dependent
The enzyme appears in viruses and cellular organisms
Synonyms
ornithine 4,5-aminomutase, d-ornithine aminomutase, d-ornithine 4,5-aminomutase, 4,5-oam, adenosylcobalamin-dependent ornithine 4,5-aminomutase, 
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SYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
adenosylcobalamin-dependent ornithine 4,5-aminomutase
Aminomutase, D-ornithine 4,5-
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D-ornithine aminomutase
OAM
oraE
oraS
ornithine 4,5-aminomutase
adenosylcobalamin-dependent ornithine 4,5-aminomutase
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adenosylcobalamin-dependent ornithine 4,5-aminomutase
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oraE
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gene name, one of the two subunits, gene is in synteny with further genes of the ornithine fermentation pathway: or-1, or-2, or-4, oraS, oraE, or-4, or-5
oraE
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gene name, one of the two subunits, gene is in synteny with further genes of the ornithine fermentation pathway: or-1, or-2, or-4, oraS, oraE, or-4, or-5
oraE
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gene name, one of the two subunits, gene is in synteny with further genes of the ornithine fermentation pathway: or-1, or-2, or-4, oraS, oraE, or-4, or-5
oraE
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gene name, one of the two subunits, gene is in synteny with further genes of the ornithine fermentation pathway: or-1, or-2, or-4, oraS, oraE, or-4, or-5
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oraE
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gene name, one of the two subunits, gene is in synteny with further genes of the ornithine fermentation pathway: or-1, or-2, or-4, oraS, oraE, or-4, or-5
oraE
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gene name, one of the two subunits, gene is in synteny with further genes of the ornithine fermentation pathway: or-1, or-2, or-4, oraS, oraE, or-4, or-5
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oraE
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gene name, one of the two subunits, gene is in synteny with further genes of the ornithine fermentation pathway: or-1, or-2, or-4, oraS, oraE, or-4, or-5
oraE
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gene name, one of the two subunits, gene is in synteny with further genes of the ornithine fermentation pathway: or-1, or-2, or-4, oraS, oraE, or-4, or-5
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oraE
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gene name, one of the two subunits, gene is in synteny with further genes of the ornithine fermentation pathway: or-1, or-2, or-4, oraS, oraE, or-4 and or-5 are placed elsewhere in the genome
oraE
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gene name, one of the two subunits, gene is in synteny with further genes of the ornithine fermentation pathway: or-1, or-2, or-4, oraS, oraE and or-4, or-5 is placed elsewhere in the genome
oraE
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gene name, one of the two subunits, gene is in synteny with further genes of the ornithine fermentation pathway: or-1, or-2, or-4, oraS, oraE and or-4, or-5 is placed elsewhere in the genome
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oraE
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gene name, one of the two subunits, gene is in synteny with further genes of the ornithine fermentation pathway: or-1, or-2, or-4, oraS, oraE, or-4 and or-5 are placed elsewhere in the genome
oraE
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gene name, one of the two subunits, gene is in synteny with further genes of the ornithine fermentation pathway: or-1, or-2, or-4, oraS, oraE, or-4 and or-5 are placed elsewhere in the genome
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oraE
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gene name, one of the two subunits, gene is in synteny with further genes of the ornithine fermentation pathway: or-1, or-2, or-4, oraS, oraE, or-4, or-5
oraE
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gene name, one of the two subunits, gene is in synteny with further genes of the ornithine fermentation pathway: or-1, or-2, or-4, oraS, oraE, or-4, or-5
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oraE
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gene name, one of the two subunits, gene is in synteny with further genes of the ornithine fermentation pathway: or-1, or-2, or-4, oraS, oraE, or-4 and or-5 are placed elsewhere in the genome
oraE
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gene name, one of the two subunits, gene is in synteny with further genes of the ornithine fermentation pathway: or-1, or-2, or-4, oraS, oraE, or-4 and or-5 are placed elsewhere in the genome
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oraS
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gene name, one of the two subunits, gene is in synteny with further genes of the ornithine fermentation pathway: or-1, or-2, or-4, oraS, oraE, or-4, or-5
oraS
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gene name, one of the two subunits, gene is in synteny with further genes of the ornithine fermentation pathway: or-1, or-2, or-4, oraS, oraE, or-4, or-5
oraS
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gene name, one of the two subunits, gene is in synteny with further genes of the ornithine fermentation pathway: or-1, or-2, or-4, oraS, oraE, or-4, or-5
oraS
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gene name, one of the two subunits, gene is in synteny with further genes of the ornithine fermentation pathway: or-1, or-2, or-4, oraS, oraE, or-4, or-5
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oraS
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gene name, one of the two subunits, gene is in synteny with further genes of the ornithine fermentation pathway: or-1, or-2, or-4, oraS, oraE, or-4, or-5
oraS
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gene name, one of the two subunits, gene is in synteny with further genes of the ornithine fermentation pathway: or-1, or-2, or-4, oraS, oraE, or-4, or-5
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oraS
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gene name, one of the two subunits, gene is in synteny with further genes of the ornithine fermentation pathway: or-1, or-2, or-4, oraS, oraE, or-4, or-5
oraS
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gene name, one of the two subunits, gene is in synteny with further genes of the ornithine fermentation pathway: or-1, or-2, or-4, oraS, oraE, or-4, or-5
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oraS
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gene name, one of the two subunits, gene is in synteny with further genes of the ornithine fermentation pathway: or-1, or-2, or-4, oraS, oraE, or-4, or-5
oraS
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gene name, one of the two subunits, gene is in synteny with further genes of the ornithine fermentation pathway: or-1, or-2, or-4, oraS, oraE, or-4, or-5
oraS
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gene name, one of the two subunits, gene is in synteny with further genes of the ornithine fermentation pathway: or-1, or-2, or-4, oraS, oraE, or-4, or-5
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oraS
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gene name, one of the two subunits, gene is in synteny with further genes of the ornithine fermentation pathway: or-1, or-2, or-4, oraS, oraE, or-4, or-5
oraS
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gene name, one of the two subunits, gene is in synteny with further genes of the ornithine fermentation pathway: or-1, or-2, or-4, oraS, oraE, or-4, or-5
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oraS
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gene name, one of the two subunits, gene is in synteny with further genes of the ornithine fermentation pathway: or-1, or-2, or-4, oraS, oraE, or-4, or-5
oraS
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gene name, one of the two subunits, gene is in synteny with further genes of the ornithine fermentation pathway: or-1, or-2, or-4, oraS, oraE, or-4, or-5
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oraS
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gene name, one of the two subunits, gene is in synteny with further genes of the ornithine fermentation pathway: or-1, or-2, or-4, oraS, oraE, or-4, or-5
oraS
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gene name, one of the two subunits, gene is in synteny with further genes of the ornithine fermentation pathway: or-1, or-2, or-4, oraS, oraE, or-4, or-5
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ornithine 4,5-aminomutase
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REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT
LITERATURE
D-ornithine = (2R,4S)-2,4-diaminopentanoate
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D-ornithine = (2R,4S)-2,4-diaminopentanoate
catalytic mechanism, overview
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D-ornithine = (2R,4S)-2,4-diaminopentanoate
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
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
radical-based catalysis mechanism, closed, active enzyme form modeling, overview
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D-ornithine = (2R,4S)-2,4-diaminopentanoate
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
D-ornithine = (2R,4S)-2,4-diaminopentanoate
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
catalytic mechanism of ornithine 4,5-aminomutase. Substrate binding results in formation of a Schiff base between the terminal amino group of the substrate and the imine nitrogen of the PLP cofactor. Subsequent homolysis of the Co-C bond generates cob(II)alamin and the highly reactive 5'-deoxyadenosyl radical, which abstracts a hydrogen atom from the C4 of the substrate. The substrate radical intermediate then rearranges to the product-like radical intermediate via a proposed cyclic intermediate. Re-abstraction of a hydrogen atom from 5'-deoxyadenosine regenerates the 5'-deoxyadenosyl radical. Product release and recombination between cob(II)alamin and the 5'-deoxyadenosyl radical completes the catalytic cycle
D-ornithine = (2R,4S)-2,4-diaminopentanoate
catalytic mechanism, detailed overview. The substrate forms a covalent Schiff base linkage with the imine nitrogen of the pyridoxal 5'-phosphate cofactor. In particular, Tyr187 forms a Pi-stacking interaction with the pyridine ring of pyridoxal 5'-phosphate, the guanidinium side chain of Arg297 forms a salt bridge with the alpha-carboxylate group of the substrate, and residues His225, His182, Asn226, Glu81, and Ser162 provide additional hydrogen bonding interactions with the substrate and cofactor
D-ornithine = (2R,4S)-2,4-diaminopentanoate
the proposed catalytic cycle of OAM starts with substrate binding, which triggers homolytic rupture of the Co-C bond to generate cob(II)alamin and the transient 5'-deoxyadenosyl radical (AdoCH2C), which subsequently abstracts a hydrogen atom from the pyridoxal 5'-phosphate-bound substrate. This results in a pyridoxal 5'-phosphate-bound substrate radical (CYC-1) that isomerises to form a pyridoxal 5'-phosphate-bound product radical (CYC+1) via a cyclic aziridinylcarbinyl intermediate (CYC). Re-abstraction of the hydrogen atom from 5'-deoxyadenosine (AdoCH3) by CYC+1 produces AdoCH2C, which recombines with cob(II)alamin to regenerate the 5'-deoxyadenosylcobalamin Co-C bond
D-ornithine = (2R,4S)-2,4-diaminopentanoate
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
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
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D-ornithine = (2R,4S)-2,4-diaminopentanoate
catalytic mechanism of ornithine 4,5-aminomutase. Substrate binding results in formation of a Schiff base between the terminal amino group of the substrate and the imine nitrogen of the PLP cofactor. Subsequent homolysis of the Co-C bond generates cob(II)alamin and the highly reactive 5'-deoxyadenosyl radical, which abstracts a hydrogen atom from the C4 of the substrate. The substrate radical intermediate then rearranges to the product-like radical intermediate via a proposed cyclic intermediate. Re-abstraction of a hydrogen atom from 5'-deoxyadenosine regenerates the 5'-deoxyadenosyl radical. Product release and recombination between cob(II)alamin and the 5'-deoxyadenosyl radical completes the catalytic cycle
-
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D-ornithine = (2R,4S)-2,4-diaminopentanoate
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
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
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REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
group transfer
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intramolecular, amino group
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isomerization
isomerization
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PATHWAY SOURCE
PATHWAYS
BRENDA
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SYSTEMATIC NAME
IUBMB Comments
D-ornithine 4,5-aminomutase
A pyridoxal-phosphate protein that requires a cobamide coenzyme for activity.
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CAS REGISTRY NUMBER
COMMENTARY
62213-30-3
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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
DL-ornithine
(2R,4S)-2,4-diaminopentanoate
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r
D-ornithine
(2R,4S)-2,4-diaminopentanoate
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-
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r
D-ornithine
(2R,4S)-2,4-diaminopentanoate
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-
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r
D-ornithine
(2R,4S)-2,4-diaminopentanoate
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second step in the oxidation pathway of L-ornithine
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r
D-ornithine
(2R,4S)-2,4-diaminopentanoate
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
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-
?
D-ornithine
(2R,4S)-2,4-diaminopentanoate
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
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-
?
D-ornithine
(2R,4S)-2,4-diaminopentanoate
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
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-
?
D-ornithine
(2R,4S)-2,4-diaminopentanoate
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the enzyme is highly specific for D-ornithine as a substrate, substrate binding structure, overview
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r
D-ornithine
(2R,4S)-2,4-diaminopentanoate
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-
-
?
D-ornithine
(2R,4S)-2,4-diaminopentanoate
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
-
-
-
?
D-ornithine
(2R,4S)-2,4-diaminopentanoate
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
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-
-
r
D-ornithine
(2R,4S)-2,4-diaminopentanoate
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
-
-
?
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
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-
?
additional information
?
-
adenosylcobalamin-dependent ornithine 4,5-aminomutase from Clostridium sticklandii utilizes pyridoxal 5'-phosphate to interconvert D-ornithine to 2,4-diaminopentanoate via a multistep mechanism that involves two hydrogen transfer steps. Important role of enzyme residue tyrosine 187, which lies planar to the pyridoxal 5'-phosphate pyridine ring. Coupled enzyme assay of ornithine 4,5-aminomutase with 2,4-diaminopentanoate dehydrogenase with DL-ornithine and DL-ornithine-3,3,4,4,5,5-d6 as substrates
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-
?
additional information
?
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adenosylcobalamin-dependent ornithine 4,5-aminomutase from Clostridium sticklandii utilizes pyridoxal 5'-phosphate to interconvert D-ornithine to 2,4-diaminopentanoate via a multistep mechanism that involves two hydrogen transfer steps. Important role of enzyme residue tyrosine 187, which lies planar to the pyridoxal 5'-phosphate pyridine ring. Coupled enzyme assay of ornithine 4,5-aminomutase with 2,4-diaminopentanoate dehydrogenase with DL-ornithine and DL-ornithine-3,3,4,4,5,5-d6 as substrates
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-
?
additional information
?
-
cobalamin-dependent enzymes enhance the rate of C-Co bond cleavage by up to 1012-fold to generate cob(II)alamin and a transient adenosyl radical. In the case of the pyridoxal 5'-phosphate and cobalamin-dependent enzymes lysine 5,6-aminomutase (EC 5.4.3.3) and ornithine 4,5 aminomutase, it has been proposed that a large scale domain reorientation of the cobalamin-binding domain is linked to radical catalysis Coupled enzyme assay with (2R,4S)-2,4-diaminopentanoate dehydrogenase (DAPDH) from Clostridium difficile
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-
?
additional information
?
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enzyme OAM is highly specific for D-ornithine, which forms non-covalent interactions with R297 and E81 through its alpha-amine and alpha-carboxylate groups. Neither wild type enzyme OAM nor any of the R297 or E81 variants are active towards the larger substrate
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-
?
additional information
?
-
-
enzyme OAM is highly specific for D-ornithine, which forms non-covalent interactions with R297 and E81 through its alpha-amine and alpha-carboxylate groups. Neither wild type enzyme OAM nor any of the R297 or E81 variants are active towards the larger substrate
-
-
?
additional information
?
-
enzyme OAM is highly specific for D-ornithine, which forms non-covalent interactions with R297 and E81 through its alpha-amine and alpha-carboxylate groups. Neither wild type enzyme OAM nor any of the R297 or E81 variants are active towards the larger substrate
-
-
?
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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
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
-
-
?
D-ornithine
(2R,4S)-2,4-diaminopentanoate
-
-
-
r
D-ornithine
(2R,4S)-2,4-diaminopentanoate
-
-
-
r
D-ornithine
(2R,4S)-2,4-diaminopentanoate
-
second step in the oxidation pathway of L-ornithine
-
r
D-ornithine
(2R,4S)-2,4-diaminopentanoate
-
-
-
?
D-ornithine
(2R,4S)-2,4-diaminopentanoate
-
-
-
?
D-ornithine
(2R,4S)-2,4-diaminopentanoate
-
-
-
r
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COFACTOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
5'-deoxyadenosylcobalamin
-
shows ability to produce highly reactive 5'-deoxyadenosyl radical in enzymatic environments
adenosylcobalamin
binding analysis with recombinant wild-type and mutant enzymes, overview
adenosylcobalamin
paramagnetic Co2+ metal center of the cob(II)alamin cofactor
pyridoxal 5'-phosphate
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
quantum mechanics/molecular mechanics (QM/MM) studies on the mechanism of action of cofactor pyridoxal 5'-phosphate in ornithine 4,5-aminomutase, overview
pyridoxal 5'-phosphate
required, important role of enzyme residue tyrosine 187, which lies planar to the pyridoxal 5'-phosphate pyridine ring. The substrate forms a covalent Schiff base linkage with the imine nitrogen of the pyridoxal 5'-phosphate cofactor. In particular, Tyr187 forms a Pi-stacking interaction with the pyridine ring of pyridoxal 5'-phosphate, the guanidinium side chain of Arg297 forms a salt bridge with the alpha-carboxylate group of the substrate, and residues His225, His182, Asn226, Glu81, and Ser162 provide additional hydrogen bonding interactions with the substrate and cofactor
pyridoxal 5'-phosphate
the protonation state of the pyridoxal 5'-phosphate cofactor has less of a role in radical-mediated chemistry compared to electrostatic interactions between the substrate and protein. Binding structure analysis and comparison with lysien 5,6-aminomutase, EC 5.4.3.3
additional information
adenosylcobalamin-dependent ornithine 4,5-aminomutase from Clostridium sticklandii utilizes pyridoxal 5'-phosphate to interconvert D-ornithine to 2,4-diaminopentanoate via a multistep mechanism that involves two hydrogen transfer steps
-
additional information
-
adenosylcobalamin-dependent ornithine 4,5-aminomutase from Clostridium sticklandii utilizes pyridoxal 5'-phosphate to interconvert D-ornithine to 2,4-diaminopentanoate via a multistep mechanism that involves two hydrogen transfer steps
-
additional information
ornithine 4,5-aminomutase is a 5'-deoxyadenosylcobalamin and pyridoxal 5'-phosphate co-dependent radical enzyme. Identification of a dynamic interface between the cobalamin and pyridoxal 5'-phosphate-binding domains, overview. Following ligand binding-induced cleavage of the Lys629-pyridoxal 5'-phosphate covalent bond, dynamic motion of the cobalamin-binding domain leads to conformational sampling of the available space. This supports radical catalysis through transient formation of a catalytically competent active state. Crucially, it appears that the formation of the state containing both a substrate/product radical and Co(II) does not restrict cobalamin domain motion
-
additional information
ornithine 4,5-aminomutase is a 5'-deoxyadenosylcobalamin and pyridoxal 5'-phosphate co-dependent radical enzyme. The conserved residues include S162 and N226, which form hydrogen bonds to the pyridine nitrogen and the phenolic group of pyridoxal 5'-phosphate, respectively. Other residues include Y187 and Y160, which flank the pyridine ring. The former aromatic side chain also forms a hydrogen bond to the pyridoxal 5'-phosphate phosphate, while the latter side chain forms a hydrogen bond to AdoCbl in a modelled closed conformation of the enzyme
-
additional information
-
ornithine 4,5-aminomutase is a 5'-deoxyadenosylcobalamin and pyridoxal 5'-phosphate co-dependent radical enzyme. The conserved residues include S162 and N226, which form hydrogen bonds to the pyridine nitrogen and the phenolic group of pyridoxal 5'-phosphate, respectively. Other residues include Y187 and Y160, which flank the pyridine ring. The former aromatic side chain also forms a hydrogen bond to the pyridoxal 5'-phosphate phosphate, while the latter side chain forms a hydrogen bond to AdoCbl in a modelled closed conformation of the enzyme
-
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METALS and IONS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
Co2+
in the paramagnetic Co2+ metal center of the cob(II)alamin cofactor
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INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
DL-2,4-diaminobutyric acid
DAB, a competitive inhibitor
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ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
mercaptan
-
keeps essential labile sulfhydryl groups reduced and protects from oxygen
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.029
D-ornithine
pH 8.5, 25°C, recombinant beta-subunit mutant S162A
0.03
D-ornithine
recombinant His-tagged mutant E338A, pH 7.5, 30°C
0.031
D-ornithine
pH 8.5, 25°C, recombinant beta-subunit mutant Y187F
0.043
D-ornithine
pH 8.5, 25°C, recombinant wild-type enzyme
0.043
D-ornithine
recombinant His-tagged mutant E338D, pH 7.5, 30°C
0.061
D-ornithine
recombinant His-tagged mutant E338Q, pH 7.5, 30°C
0.12
D-ornithine
pH 8.5, 25°C, recombinant beta-subunit mutant N226D
0.14
D-ornithine
recombinant mutant H225A, pH 8.5, 25°C
0.14
D-ornithine
pH 8.5, 25°C, recombinant beta-subunit mutant Y160F
0.176
D-ornithine
pH 8.5, 25°C, recombinant beta-subunit mutant E338A
0.185
D-ornithine
pH 8.5, 25°C, recombinant beta-subunit mutant I424E
0.186
D-ornithine
pH 8.5, 25°C, recombinant beta-subunit mutant C700S
0.189
D-ornithine
pH 8.5, 25°C, recombinant wild-type enzyme
0.19
D-ornithine
recombinant His-tagged wild-type enzyme, pH 7.5, 30°C
0.19
D-ornithine
recombinant wild-type, pH 8.5, 25°C
0.19
D-ornithine
pH 8.5, 25°C, recombinant beta-subunit mutant G339W
0.193
D-ornithine
pH 8.5, 25°C, recombinant beta-subunit mutant D627A
0.193
D-ornithine
pH 8.5, 25°C, recombinant beta-subunit mutant P343W
0.2
D-ornithine
-
pH and temperature not specified in the publication
0.453
D-ornithine
recombinant mutant H225Q, pH 8.5, 25°C
0.78
D-ornithine
pH 8.5, 25°C, recombinant beta-subunit mutant E81A
0.87
D-ornithine
pH 8.5, 25°C, recombinant beta-subunit mutant E81Q
4.9
D-ornithine
pH 8.5, 25°C, recombinant beta-subunit mutant R297K
0.068
DL-Ornithine
pH 8.5, 20°C, recombinant beta-subunit mutant Y187A
0.162
DL-Ornithine
pH 8.5, 20°C, recombinant beta-subunit mutant Y187F
0.567
DL-Ornithine
pH 8.5, 20°C, recombinant wild-type enzyme
additional information
additional information
Michaelis-Menten kinetics
-
additional information
additional information
Michaelis-Menten kinetics
-
additional information
additional information
pre-steady-state and stedy-state kinetics of wild-type and mutant enzymes
-
additional information
additional information
pre-steady-state and stedy-state kinetics of wild-type and mutant enzymes
-
additional information
additional information
-
pre-steady-state and stedy-state kinetics of wild-type and mutant enzymes
-
additional information
additional information
kinetic isotope effects analysis using isotope-labeled DL-ornithine-3,3,4,4,5,5-d6 revealing a diminished Dkcat/Km of 2.5 relative to a Dkcat of 7.6, suggesting slow release of the substrate from the active site. Kinetic isotope effects are not observed on the he rate constant associated with Co-C bond homolysis as this step is likely gated by the formation of the external aldimine. Stopped-flow kinetics, and Michaelis-Menten steady-state kinetics
-
additional information
additional information
-
kinetic isotope effects analysis using isotope-labeled DL-ornithine-3,3,4,4,5,5-d6 revealing a diminished Dkcat/Km of 2.5 relative to a Dkcat of 7.6, suggesting slow release of the substrate from the active site. Kinetic isotope effects are not observed on the he rate constant associated with Co-C bond homolysis as this step is likely gated by the formation of the external aldimine. Stopped-flow kinetics, and Michaelis-Menten steady-state kinetics
-
additional information
additional information
steady-state and pre-steady-state kinetics
-
additional information
additional information
steady-state kinetics of wild-type enzyme and beta-subunit mutants, enzyme dynamics and C-Co bond homolysis in single-turnover stopped-flow kinetics
-
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.002
D-ornithine
pH 8.5, 25°C, recombinant beta-subunit mutant E81D
0.013
D-ornithine
pH 8.5, 25°C, recombinant beta-subunit mutant E81A
0.027
D-ornithine
pH 8.5, 25°C, recombinant beta-subunit mutant Y160F
0.029
D-ornithine
pH 8.5, 25°C, recombinant beta-subunit mutant E81Q
0.032
D-ornithine
recombinant His-tagged mutant E338D, pH 7.5, 30°C
0.068
D-ornithine
pH 8.5, 25°C, recombinant beta-subunit mutant R297K
0.12
D-ornithine
pH 8.5, 25°C, recombinant beta-subunit mutant Y187F
0.14
D-ornithine
pH 8.5, 25°C, recombinant beta-subunit mutant P343W
0.2
D-ornithine
pH 8.5, 25°C, recombinant beta-subunit mutant G339W
0.24
D-ornithine
pH 8.5, 25°C, recombinant beta-subunit mutant E338A
0.3
D-ornithine
recombinant mutant H225A, pH 8.5, 25°C
0.37
D-ornithine
pH 8.5, 25°C, recombinant beta-subunit mutant S162A
0.38
D-ornithine
pH 8.5, 25°C, recombinant beta-subunit mutant N226D
0.76
D-ornithine
pH 8.5, 25°C, recombinant beta-subunit mutant I424E
1
D-ornithine
recombinant mutant H225Q, pH 8.5, 25°C
2.88
D-ornithine
pH 8.5, 25°C, recombinant beta-subunit mutant C700S
2.89
D-ornithine
pH 8.5, 25°C, recombinant beta-subunit mutant D627A
2.9
D-ornithine
recombinant His-tagged wild-type enzyme, pH 7.5, 30°C
2.9
D-ornithine
recombinant wild-type, pH 8.5, 25°C
2.97
D-ornithine
pH 8.5, 25°C, recombinant wild-type enzyme
4.3
D-ornithine
recombinant His-tagged mutant E338Q, pH 7.5, 30°C
7.6
D-ornithine
recombinant His-tagged mutant E338A, pH 7.5, 30°C
8.2
D-ornithine
pH 8.5, 25°C, recombinant wild-type enzyme
564
D-ornithine
recombinant beta-subunit mutant E338A, pH 8.5, 25°C
608
D-ornithine
recombinant beta-subunit mutant E338D, pH 8.5, 25°C
694
D-ornithine
recombinant beta-subunit mutant E338Q, pH 8.5, 25°C
1072
D-ornithine
recombinant wild-type enzyme, pH 8.5, 25°C
0.0023
DL-Ornithine
pH 8.5, 20°C, recombinant beta-subunit mutant Y187A
0.115
DL-Ornithine
pH 8.5, 20°C, recombinant beta-subunit mutant Y187F
2.9
DL-Ornithine
pH 8.5, 20°C, recombinant wild-type enzyme
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kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.014
D-ornithine
pH 8.5, 25°C, recombinant beta-subunit mutant R297K
0.016
D-ornithine
pH 8.5, 25°C, recombinant beta-subunit mutant E81A
0.07
D-ornithine
recombinant His-tagged mutant E338Q, pH 7.5, 30°C
0.19
D-ornithine
pH 8.5, 25°C, recombinant beta-subunit mutant Y160F
0.25
D-ornithine
recombinant His-tagged mutant E338A, pH 7.5, 30°C
0.33
D-ornithine
pH 8.5, 25°C, recombinant beta-subunit mutant E81Q
0.72
D-ornithine
pH 8.5, 25°C, recombinant beta-subunit mutant P343W
0.75
D-ornithine
recombinant His-tagged mutant E338D, pH 7.5, 30°C
1.05
D-ornithine
pH 8.5, 25°C, recombinant beta-subunit mutant G339W
1.36
D-ornithine
pH 8.5, 25°C, recombinant beta-subunit mutant E338A
2.2
D-ornithine
recombinant mutant H225A, pH 8.5, 25°C
2.2
D-ornithine
recombinant mutant H225Q, pH 8.5, 25°C
3.2
D-ornithine
pH 8.5, 25°C, recombinant beta-subunit mutant N226D
3.9
D-ornithine
pH 8.5, 25°C, recombinant beta-subunit mutant Y187F
4.11
D-ornithine
pH 8.5, 25°C, recombinant beta-subunit mutant I424E
13
D-ornithine
pH 8.5, 25°C, recombinant beta-subunit mutant S162A
14.97
D-ornithine
pH 8.5, 25°C, recombinant beta-subunit mutant D627A
15.2
D-ornithine
-
pH and temperature not specified in the publication
15.2
D-ornithine
recombinant His-tagged wild-type enzyme, pH 7.5, 30°C
15.2
D-ornithine
recombinant wild-type, pH 8.5, 25°C
15.48
D-ornithine
pH 8.5, 25°C, recombinant beta-subunit mutant C700S
15.71
D-ornithine
pH 8.5, 25°C, recombinant wild-type enzyme
190
D-ornithine
pH 8.5, 25°C, recombinant wild-type enzyme
0.034
DL-Ornithine
pH 8.5, 20°C, recombinant beta-subunit mutant Y187A
0.71
DL-Ornithine
pH 8.5, 20°C, recombinant beta-subunit mutant Y187F
5.11
DL-Ornithine
pH 8.5, 20°C, recombinant wild-type enzyme
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Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.0046
2,4-diaminobutyrate
pH 8.5, 25°C, recombinant wild-type enzyme
0.032
2,4-diaminobutyrate
pH 8.5, 25°C, recombinant mutant Y187F
0.1
2,4-diaminobutyrate
pH 8.5, 25°C, recombinant mutant N226D
0.27
2,4-diaminobutyrate
pH 8.5, 25°C, recombinant mutant S162A
2
2,4-diaminobutyrate
pH 8.5, 25°C, recombinant mutant Y160F
0.0046
2,4-diaminobutyric acid
recombinant wild-type, pH 8.5, 25°C
0.095
2,4-diaminobutyric acid
recombinant mutant H225A, pH 8.5, 25°C
0.098
2,4-diaminobutyric acid
recombinant mutant H225Q, pH 8.5, 25°C
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SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
8.5
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pH RANGE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
25
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TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
subunits beta and alpha
UniProt
brenda
subunits beta and alpha; i.e. Clostridium sticklandii
UniProt
brenda
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GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
evolution
-
the enzyme belongs to the class III dAdoCbl-dependent isomerase family
malfunction
metabolism
additional information
malfunction
conservative substitutions of the residues that form salt bridges to the alpha-carboxylate (R297) or the alpha-amine (E81) of D-ornithine results in a 300-600fold reduction in catalytic turnover and a more pronounced 1000 to 14000fold decrease in catalytic efficiency compared to wild-type. Mutating residues that solely interact with the pyridoxal 5'-phosphate cofactor leads to more modest decreases (10-60fold) in kcat and kcat/Km. All but one variant (S162A) elicite an increase in the kinetic isotope effect on kcat and kcat/Km with D,L-ornithine-3,3,4,4,5,5-d6 as the substrate, which indicates that hydrogen atom abstraction is more rate determining. The substitutions decrease the extent of CoeC bond homolysis, they do not affect the structural integrity of the active site
malfunction
substrate-induced C-Co bond homolysis is compromised in Glu388 variant forms of OAM, although photolysis of the C-Co bond is not affected by the identity of residue 338. Electrostatic interactions of Glu338 with the 5'-deoxyadenosyl group of B12 potentiate C-Co bond homolysis in closed conformations only. These conformations are unlocked by substrate binding. Conformational sampling of the mobile cobalamin-binding domain occurs in the Glu388 variants, but that, in adopting the closed conformation, C-Co bond homolysis is compromised. The population of the closed conformation has an associated lifetime. Each time the closed conformation is populated, there is a higher probability of bond homolysis in wild-type OAM relative to the variant forms because of the electrostatic interactions formed between Glu338 and the cobalamin cofactor that facilitate bond homolysis
malfunction
-
conservative substitutions of the residues that form salt bridges to the alpha-carboxylate (R297) or the alpha-amine (E81) of D-ornithine results in a 300-600fold reduction in catalytic turnover and a more pronounced 1000 to 14000fold decrease in catalytic efficiency compared to wild-type. Mutating residues that solely interact with the pyridoxal 5'-phosphate cofactor leads to more modest decreases (10-60fold) in kcat and kcat/Km. All but one variant (S162A) elicite an increase in the kinetic isotope effect on kcat and kcat/Km with D,L-ornithine-3,3,4,4,5,5-d6 as the substrate, which indicates that hydrogen atom abstraction is more rate determining. The substitutions decrease the extent of CoeC bond homolysis, they do not affect the structural integrity of the active site
-
metabolism
-
catalyzes step 2 in the ornithine fermentation pathway
metabolism
-
catalyzes step 2 in the ornithine fermentation pathway
metabolism
-
catalyzes step 2 in the ornithine fermentation pathway
metabolism
-
catalyzes step 2 in the ornithine fermentation pathway
metabolism
-
catalyzes step 2 in the ornithine fermentation pathway
metabolism
-
catalyzes step 2 in the ornithine fermentation pathway
metabolism
the enzyme catalyzes the second step in the oxidative breakdown of the amino acid, converting D-ornithine to 2,4-diaminopentanoic acid
metabolism
-
catalyzes step 2 in the ornithine fermentation pathway
-
metabolism
-
the enzyme catalyzes the second step in the oxidative breakdown of the amino acid, converting D-ornithine to 2,4-diaminopentanoic acid
-
metabolism
-
catalyzes step 2 in the ornithine fermentation pathway
-
metabolism
-
catalyzes step 2 in the ornithine fermentation pathway
-
metabolism
-
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
additional information
computational study on the experimentally elusive cyclisation step in the cofactor pyridoxal 5'-phosphate-dependent D-ornithine 4,5-aminomutase-catalysed reaction, quantum mechanics/molecular mechanics (QM/MM) studies on the mechanism of action of cofactor pyridoxal 5-phosphate in ornithine 4,5-aminomutase, modeling, overview. Calculations using both model systems and a combined QM/MM approach suggest that regulation of the cyclic radical intermediate is achieved through the synergy of the intrinsic catalytic power of cofactor pyridoxal 5'-phosphate and enzyme active site. The captodative effect of pyridoxal 5'-phosphate is balanced by an enzyme active site that controls the deprotonation of both the pyridine nitrogen atom (N1) and the Schiff-base nitrogen atom (N2). Electrostatic interactions between the terminal carboxylate and amino groups of the substrate and Arg297 and Glu81 impose substantial strain energy on the orientation of the cyclic intermediate to control its trajectory. In addition the strain energy, which appears to be sensitive to both the number of carbon atoms in the substrate/analogue and the position of the radical intermediates, may play a key role in controlling the transition of the enzyme from the closed to the open state
additional information
for the pyridoxal 5'-phosphate and cobalamin-dependent enzyme ornithine 4,5-aminomutase, large-scale re-orientation of the cobalamin-binding domain linked to C-Co bond breakage is proposed. In the model, substrate binding triggers dynamic sampling of the B12-binding Rossmann domain to achieve a catalytically competent closed conformational state. In closed conformations of the enzyme, Glu338 is thought to facilitate C-Co bond breakage by close association with the cobalamin adenosyl group. Large-scale motion is required to pre-organize the active site by enabling transient formation of closed conformations of OAM. In closed conformations, Glu338 interacts with the 5'-deoxyadenosyl group of cobalamin. This interaction is required to potentiate C-Co homolysis, and is a crucial component of the approximately 1012 rate enhancement achieved by cobalamin-dependent enzymes for C-Co bond homolysis. Three-dimensional enzyme structure and active site structure analysis, spectral analysis of substrate-bound wild-type and beta-subunit mutant enzymes, overview
additional information
important role in catalysis of enzyme residue tyrosine 187, which lies planar to the pyridoxal 5'-phosphate pyridine ring. The level of protein-substrate interactions in aminomutases not only influences substrate specificity, but also controls radical chemistry. Substrate molecular docking simulations
additional information
-
important role in catalysis of enzyme residue tyrosine 187, which lies planar to the pyridoxal 5'-phosphate pyridine ring. The level of protein-substrate interactions in aminomutases not only influences substrate specificity, but also controls radical chemistry. Substrate molecular docking simulations
additional information
ornithine 4,5-aminomutase (OAM) from Clostridium sticklandii is an adenosylcobalamin (AdoCbl) and pyridoxal 5'-phosphate (PLP)-dependent enzyme that catalyzes a 1,2-amino shift of the delta-amino group of Dornithine to form 2S,4R-diaminopentanoate, interconverting D-ornithine and 2S,4R-diaminopentanoate. The reaction occurs via a radical-based mechanism whereby a PLP-bound substrate radical undergoes intramolecular isomerization via an azacyclopropylcarbinyl radical intermediate. Analysis of the catalytic role of active site residues that form non-covalent interactions with PLP and/or substrate, D-ornithine, and kinetic analysis, overview. Residues that form salt bridges to the alpha-carboxylate (R297) or the alpha-amine (E81) of D-ornithine are most critical for the enzyme activity. The protonation state of the pyridoxal 5'-phosphate cofactor has less of a role in radical-mediated chemistry compared to electrostatic interactions between the substrate and protein. Active site of OAM (PDB ID 3KOZ) with bound substrate D-ornithine in the modelled closed conformation, overview
additional information
-
ornithine 4,5-aminomutase (OAM) from Clostridium sticklandii is an adenosylcobalamin (AdoCbl) and pyridoxal 5'-phosphate (PLP)-dependent enzyme that catalyzes a 1,2-amino shift of the delta-amino group of Dornithine to form 2S,4R-diaminopentanoate, interconverting D-ornithine and 2S,4R-diaminopentanoate. The reaction occurs via a radical-based mechanism whereby a PLP-bound substrate radical undergoes intramolecular isomerization via an azacyclopropylcarbinyl radical intermediate. Analysis of the catalytic role of active site residues that form non-covalent interactions with PLP and/or substrate, D-ornithine, and kinetic analysis, overview. Residues that form salt bridges to the alpha-carboxylate (R297) or the alpha-amine (E81) of D-ornithine are most critical for the enzyme activity. The protonation state of the pyridoxal 5'-phosphate cofactor has less of a role in radical-mediated chemistry compared to electrostatic interactions between the substrate and protein. Active site of OAM (PDB ID 3KOZ) with bound substrate D-ornithine in the modelled closed conformation, overview
additional information
-
ornithine 4,5-aminomutase (OAM) from Clostridium sticklandii is an adenosylcobalamin (AdoCbl) and pyridoxal 5'-phosphate (PLP)-dependent enzyme that catalyzes a 1,2-amino shift of the delta-amino group of Dornithine to form 2S,4R-diaminopentanoate, interconverting D-ornithine and 2S,4R-diaminopentanoate. The reaction occurs via a radical-based mechanism whereby a PLP-bound substrate radical undergoes intramolecular isomerization via an azacyclopropylcarbinyl radical intermediate. Analysis of the catalytic role of active site residues that form non-covalent interactions with PLP and/or substrate, D-ornithine, and kinetic analysis, overview. Residues that form salt bridges to the alpha-carboxylate (R297) or the alpha-amine (E81) of D-ornithine are most critical for the enzyme activity. The protonation state of the pyridoxal 5'-phosphate cofactor has less of a role in radical-mediated chemistry compared to electrostatic interactions between the substrate and protein. Active site of OAM (PDB ID 3KOZ) with bound substrate D-ornithine in the modelled closed conformation, overview
-
Show AA Sequence and Transmembrane Helices (126 entries)
Please use the AA Sequence and Transmembrane Helices Search for a specific query.
Please use the AA Sequence and Transmembrane Helices Search for a specific query.
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MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
12800
180000
-
gel electrophoresis, gel filtration, sucrose density gradient centrifugation
82900
90000
-
2 * 12800, subunit OraS + 2 * 90000, subunit OraE, alpha2beta2
12800
-
alpha2beta2, 2 * 12800 + 2 * 82900, deduced from nucleotide sequence
12800
alpha2beta2, OraS, OraE, 2 * 12800 + 2 * 82900
12800
2 * 12800, subunit OraS + 2 * 82900, subunit OraE, alpha2beta2
12800
-
2 * 12800, subunit OraS + 2 * 90000, subunit OraE, alpha2beta2
82900
-
alpha2beta2, 2 * 12800 + 2 * 82900, deduced from nucleotide sequence
82900
alpha2beta2, OraS, OraE, 2 * 12800 + 2 * 82900
82900
2 * 12800, subunit OraS + 2 * 82900, subunit OraE, alpha2beta2
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SUBUNIT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
heterotetramer
tetramer
additional information
heterotetramer
alpha2beta2, OraS, OraE, 2 * 12800 + 2 * 82900
heterotetramer
alpha2beta2, 2 * 12800, alpha-subunit, + 2 * 82900, beta-subunit, 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
-
2 * 12800, subunit OraS + 2 * 82900, subunit OraE, alpha2beta2
-
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
additional information
enzyme OAM is an alpha2beta2 heterodimer comprised of two strongly associating subunits, OraE (82.9 kDa) and OraS (12.8 kDa).7 Each OraE subunit comprises a triosephosphate isomerase (TIM) barrel domain and a Rossmann-like domain. A domain swap occurs in the heterodimer, whereby the Rossmann-like domain of one OraE subunit associates with the TIM barrel domain of a second OraE subunit and vice versa, such that two identical active sites are formed
additional information
-
enzyme OAM is an alpha2beta2 heterodimer comprised of two strongly associating subunits, OraE (82.9 kDa) and OraS (12.8 kDa).7 Each OraE subunit comprises a triosephosphate isomerase (TIM) barrel domain and a Rossmann-like domain. A domain swap occurs in the heterodimer, whereby the Rossmann-like domain of one OraE subunit associates with the TIM barrel domain of a second OraE subunit and vice versa, such that two identical active sites are formed
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CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
analysis of the crystal structure of the active site of ornithine 4,5-aminomutase complexed with the natural substrate D-ornithine, PDB ID 3KOZ
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
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PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
C700S
site-directed mutagenesis, the beta-subunit mutant shows similar kinetics and activity as the wild-type enzyme
D627A
site-directed mutagenesis, the beta-subunit mutant shows similar kinetics and slightly reduced activity compared to the wild-type enzyme
E338A
E338D
E338Q
E81A
site-directed mutagenesis, inactive beta-subunit mutant
E81D
site-directed mutagenesis, almost inactive beta-subunit mutant
E81Q
site-directed mutagenesis, the beta-subunit mutant shows highly reduced activity compared to wild-type
G128D
site-directed mutagenesis, inactive beta-subunit mutant
G339W
site-directed mutagenesis, the beta-subunit mutant shows altered kinetics and highly reduced activity compared to the wild-type enzyme
I424E
site-directed mutagenesis, the beta-subunit mutant shows altered kinetics and reduced activity compared to the wild-type enzyme
N226D
site-directed mutagenesis, the beta-subunit mutant shows highly reduced activity compared to wild-type
P343W
site-directed mutagenesis, the beta-subunit mutant shows altered kinetics and highly reduced activity compared to the wild-type enzyme
R297K
site-directed mutagenesis, almost inactive beta-subunit mutant
S162A
site-directed mutagenesis, the beta-subunit mutant shows highly reduced activity compared to wild-type
Y160F
site-directed mutagenesis, almost inactive beta-subunit mutant
Y187A
site-directed mutagenesis, the beta-subunit mutant shows 1260fold reduced activity, compared to wild-type, attributed to a slower rate of external aldimine formation and a diminution of adenosylcobalamin Co-C bond homolysis
Y187F
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
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
-
E81A
-
site-directed mutagenesis, inactive beta-subunit mutant
-
E81D
-
site-directed mutagenesis, almost inactive beta-subunit mutant
-
E81Q
-
site-directed mutagenesis, the beta-subunit mutant shows highly reduced activity compared to wild-type
-
S162A
-
site-directed mutagenesis, the beta-subunit mutant shows highly reduced activity compared to wild-type
-
Y160F
-
site-directed mutagenesis, almost inactive beta-subunit mutant
-
additional information
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
E338A
site-directed mutagenesis, the beta-subunit mutant shows a reduced turnover number compared to wild-type enzyme, while the Km value is similar
E338A
site-directed mutagenesis, the beta-subunit mutant shows altered kinetics and highly reduced activity compared to the wild-type enzyme
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
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 no detectable adenosylcobalamin homolysis upon binding of the physiological substrate
E338D
site-directed mutagenesis, the beta-subunit mutant shows a reduced turnover number compared to wild-type enzyme, while the Km value is similar
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
E338Q
site-directed mutagenesis, the beta-subunit mutant shows a reduced turnover number compared to wild-type enzyme, while the Km value is similar
Y187F
site-directed mutagenesis, the beta-subunit mutant shows 25fold reduced activity, compared to wild-type, attributed to a slower rate of external aldimine formation and a diminution of adenosylcobalamin Co-C bond homolysis. In the case of beta-subunit mutant Y187F, the integrity of the active site is maintained as cob(II)alamin and the pyridoxal 5'-phosphate organic radical (even at lower concentrations) remain tightly exchange-coupled
Y187F
site-directed mutagenesis, the beta-subunit mutant shows highly reduced activity compared to wild-type
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
-
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 no detectable adenosylcobalamin homolysis upon binding of the physiological substrate
-
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. OraS protein alone lowers the Km of KamDE for adenosylcobalamin and pyridoxal phosphate
additional information
analysis of substrate-bound wild-type and beta-subunit mutant enzymes, overview
additional information
OAM variants are designed to perturb the interface between the cobalamin-binding domain and the pyridoxal 5'-phosphate-binding TIM-barrel domain. Steady-state and single turnover kinetic studies of these variants, combined with pulsed electron-electron double resonance measurements of spin-labeled OAM are used to provide direct evidence for a dynamic interface between the cobalamin and pyridoxal 5'-phosphate-binding domains, overview
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TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
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STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-20°C, at a concentration of 0.7 mg/ml, 35% loss of activity after 1 month
-
-80°C, the purified enzyme can be stored in concentrated solution in the presence of 50% glycerol for several months
-
4°C, 20% glycerol v/v or 0.02 mM coenzyme B12, enzyme concentration 1.0 mg/ml, 9% loss of activity after 2 d
-
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PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
Ni2+ -NTA column chromatography and Q-Sepharose resin chromatography
-
recombinant His-tagged wild-type and beta-subunit mutant enzymes from Escherichia coli
recombinant wild-type and beta-subunit mutant enzymes
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CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
expression of oraA and oraE genes encoding D-ornithine aminomutase in Escherichia coli
-
genes oraE and oraS for subunits OraS and OraE, expression in Escherichia coli
-
genes oraS and oraE encode the alpha- and beta-subunit, respectively
genes oraS and oraE encode the alpha- and beta-subunit, respectively, recombinant expression of His-tagged wild-type and beta-subunit mutant enzymes in Escherichia coli strain XL-1 Blue
genes oraS and oraE encoding the alpha- and beta-subunit, recombinant expression of wild-type and beta-subunit mutant enzymes
recombinant expression of wild-type and beta-subunit mutant enzymes
recombinant expression of wild-type and mutant enzymes
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REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Somack, R.; Costilow, R.N.
Purification and properties of a pyridoxal phosphate and coenzyme B12 dependent D-alpha-ornithine 5,4-aminomutase
Biochemistry
12
2597-2604
1973
Acetoanaerobium sticklandii
brenda
Chen, H.P.; Wu, S.H.; Lin, Y.L.; Chen, C.M.; Tsay, S.S.
Cloning, sequencing, heterologous expression, purification, and characterization of adenosylcobalamin-dependent D-ornithine aminomutase from Clostridium sticklandii
J. Biol. Chem.
276
44744-44750
2001
Acetoanaerobium sticklandii
brenda
Chen, H.P.; Hsui, F.C.; Lin, L.Y.; Ren, C.T.; Wu, S.H.
Coexpression, purification and characterization of the E and S subunits of coenzyme B(12) and B(6) dependent Clostridium sticklandii D-ornithine aminomutase in Escherichia coli
Eur. J. Biochem.
271
4293-4297
2004
Acetoanaerobium sticklandii
brenda
Tseng, C.H.; Yang, C.H.; Lin, H.J.; Wu, C.; Chen, H.P.
The S subunit of D-ornithine aminomutase from Clostridium sticklandii is responsible for the allosteric regulation in D-alpha-lysine aminomutase
FEMS Microbiol. Lett.
274
148-153
2007
Acetoanaerobium sticklandii
brenda
Wolthers, K.R.; Rigby, S.E.; Scrutton, N.S.
Mechanism of radical-based catalysis in the reaction catalyzed by adenosylcobalamin-dependent ornithine 4,5-aminomutase
J. Biol. Chem.
283
34615-34625
2008
Acetoanaerobium sticklandii
brenda
Fonknechten, N.; Perret, A.; Perchat, N.; Tricot, S.; Lechaplais, C.; Vallenet, D.; Vergne, C.; Zaparucha, A.; Le Paslier, D.; Weissenbach, J.; Salanoubat, M.
A conserved gene cluster rules anaerobic oxidative degradation of L-ornithine
J. Bacteriol.
191
3162-3167
2009
Clostridioides difficile, Acetoanaerobium sticklandii, Cutibacterium acnes, Alkaliphilus metalliredigens, Fervidobacterium nodosum Rt17-B1, Alkaliphilus oremlandii, Petrotoga mobilis, Thermoanaerobacter pseudethanolicus, Natranaerobius thermophilus, Thermosipho melanesiensis, Natranaerobius thermophilus JW/NM-WN-LF, Clostridioides difficile 630, Thermosipho melanesiensis BI429, Petrotoga mobilis SJ95, Thermoanaerobacter pseudethanolicus MB4, Cutibacterium acnes KPA171202, Alkaliphilus oremlandii OhlLAs
brenda
Wolthers, K.R.; Levy, C.; Scrutton, N.S.; Leys, D.
Large-scale domain dynamics and adenosylcobalamin reorientation orchestrate radical catalysis in ornithine 4,5-aminomutase
J. Biol. Chem.
285
13942-13950
2010
Acetoanaerobium sticklandii (E3PY95), Acetoanaerobium sticklandii (E3PY96), Acetoanaerobium sticklandii
brenda
Makins, C.; Pickering, A.V.; Mariani, C.; Wolthers, K.R.
Mutagenesis of a conserved glutamate reveals the contribution of electrostatic energy to adenosylcobalamin co-C bond homolysis in ornithine 4,5-aminomutase and methylmalonyl-CoA mutase
Biochemistry
52
878-888
2013
Acetoanaerobium sticklandii (E3PY95), Acetoanaerobium sticklandii (E3PY96), Acetoanaerobium sticklandii, Acetoanaerobium sticklandii DSM 519 (E3PY95), Acetoanaerobium sticklandii DSM 519 (E3PY96)
brenda
Makins, C.; Miros, F.N.; Scrutton, N.S.; Wolthers, K.R.
Role of histidine 225 in adenosylcobalamin-dependent ornithine 4,5-aminomutase
Bioorg. Chem.
40
39-47
2012
Acetoanaerobium sticklandii (E3PY95), Acetoanaerobium sticklandii (E3PY96), Acetoanaerobium sticklandii DSM 519 (E3PY95), Acetoanaerobium sticklandii DSM 519 (E3PY96)
brenda
Maity, A.N.; Chen, Y.H.; Ke, S.C.
Large-scale domain motions and pyridoxal-5-phosphate assisted radical catalysis in coenzyme B12-dependent aminomutases
Int. J. Mol. Sci.
15
3064-3087
2014
Acetoanaerobium sticklandii
brenda
Pang, J.; Li, X.; Morokuma, K.; Scrutton, N.S.; Sutcliffe, M.J.
Large-scale domain conformational change is coupled to the activation of the Co-C bond in the B12-dependent enzyme ornithine 4,5-aminomutase: a computational study
J. Am. Chem. Soc.
134
2367-2377
2012
Acetoanaerobium sticklandii
brenda
Makins, C.; Whitelaw, D.A.; Mu, C.; Walsby, C.J.; Wolthers, K.R.
Isotope effects for deuterium transfer and mutagenesis of Tyr187 provide insight into controlled radical chemistry in adenosylcobalamin-dependent ornithine 4,5-aminomutase
Biochemistry
53
5432-5443
2014
Acetoanaerobium sticklandii (E3PY95 AND E3PY96), Acetoanaerobium sticklandii
brenda
Makins, C.; Whitelaw, D.A.; McGregor, M.; Petit, A.; Mothersole, R.G.; Prosser, K.E.; Wolthers, K.R.
Optimal electrostatic interactions between substrate and protein are essential for radical chemistry in ornithine 4,5-aminomutase
Biochim. Biophys. Acta
1865
1077-1084
2017
Acetoanaerobium sticklandii (E3PY95 AND E3PY96), Acetoanaerobium sticklandii, Acetoanaerobium sticklandii DSM 519 (E3PY95 AND E3PY96)
brenda
Pang, J.; Scrutton, N.S.; Sutcliffe, M.J.
Quantum mechanics/molecular mechanics studies on the mechanism of action of cofactor pyridoxal 5-phosphate in ornithine 4,5-aminomutase
Chemistry
20
11390-11401
2014
Acetoanaerobium sticklandii (E3PY95 AND E3PY96)
brenda
Menon, B.R.; Menon, N.; Fisher, K.; Rigby, S.E.; Leys, D.; Scrutton, N.S.
Glutamate 338 is an electrostatic facilitator of C-Co bond breakage in a dynamic/electrostatic model of catalysis by ornithine aminomutase
FEBS J.
282
1242-1255
2015
Acetoanaerobium sticklandii (E3PY95 AND E3PY96)
brenda
Menon, B.R.; Fisher, K.; Rigby, S.E.; Scrutton, N.S.; Leys, D.
A conformational sampling model for radical catalysis in pyridoxal phosphate- and cobalamin-dependent enzymes
J. Biol. Chem.
289
34161-34174
2014
Acetoanaerobium sticklandii (E3PY95 AND E3PY96)
brenda
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