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Literature summary extracted from

  • 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 (2017), Biochim. Biophys. Acta, 1865, 1077-1084 .
    View publication on PubMed

Cloned(Commentary)

EC Number Cloned (Comment) Organism
5.4.3.3 genes FN1863 and FN1862 encoding the alpha- and beta-subunit, respectively, recombinant expression in Escherichia coli strain Rosetta(DE3) pLysS Fusobacterium nucleatum
5.4.3.5 genes oraS and oraE encode the alpha- and beta-subunit, respectively Acetoanaerobium sticklandii

Protein Variants

EC Number Protein Variants Comment Organism
5.4.3.5 E81A site-directed mutagenesis, inactive beta-subunit mutant Acetoanaerobium sticklandii
5.4.3.5 E81D site-directed mutagenesis, almost inactive beta-subunit mutant Acetoanaerobium sticklandii
5.4.3.5 E81Q site-directed mutagenesis, the beta-subunit mutant shows highly reduced activity compared to wild-type Acetoanaerobium sticklandii
5.4.3.5 N226D site-directed mutagenesis, the beta-subunit mutant shows highly reduced activity compared to wild-type Acetoanaerobium sticklandii
5.4.3.5 R297K site-directed mutagenesis, almost inactive beta-subunit mutant Acetoanaerobium sticklandii
5.4.3.5 S162A site-directed mutagenesis, the beta-subunit mutant shows highly reduced activity compared to wild-type Acetoanaerobium sticklandii
5.4.3.5 Y160F site-directed mutagenesis, almost inactive beta-subunit mutant Acetoanaerobium sticklandii
5.4.3.5 Y187F site-directed mutagenesis, the beta-subunit mutant shows highly reduced activity compared to wild-type Acetoanaerobium sticklandii

Inhibitors

EC Number Inhibitors Comment Organism Structure
5.4.3.5 2,4-diaminobutyrate
-
 Acetoanaerobium sticklandii

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
5.4.3.5 0.029
-
 D-ornithine pH 8.5, 25°C, recombinant beta-subunit mutant S162A Acetoanaerobium sticklandii
5.4.3.5 0.031
-
 D-ornithine pH 8.5, 25°C, recombinant beta-subunit mutant Y187F Acetoanaerobium sticklandii
5.4.3.5 0.043
-
 D-ornithine pH 8.5, 25°C, recombinant wild-type enzyme Acetoanaerobium sticklandii
5.4.3.5 0.12
-
 D-ornithine pH 8.5, 25°C, recombinant beta-subunit mutant N226D Acetoanaerobium sticklandii
5.4.3.5 0.14
-
 D-ornithine pH 8.5, 25°C, recombinant beta-subunit mutant Y160F Acetoanaerobium sticklandii
5.4.3.5 0.78
-
 D-ornithine pH 8.5, 25°C, recombinant beta-subunit mutant E81A Acetoanaerobium sticklandii
5.4.3.5 0.87
-
 D-ornithine pH 8.5, 25°C, recombinant beta-subunit mutant E81Q Acetoanaerobium sticklandii
5.4.3.5 4.9
-
 D-ornithine pH 8.5, 25°C, recombinant beta-subunit mutant R297K Acetoanaerobium sticklandii

Molecular Weight [Da]

EC Number Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
5.4.3.5 191400
-
-
 Acetoanaerobium sticklandii

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
5.4.3.3 (3S)-3,6-diaminohexanoate Fusobacterium nucleatum
-
 (3S,5S)-3,5-diaminohexanoate
-
 r
5.4.3.3 D-lysine Fusobacterium nucleatum
-
 2,5-diaminohexanoate
-
 r
5.4.3.5 D-ornithine Acetoanaerobium sticklandii
-
 (2R,4S)-2,4-diaminopentanoate
-
 r
5.4.3.5 D-ornithine Acetoanaerobium sticklandii DSM 519
-
 (2R,4S)-2,4-diaminopentanoate
-
 r

Organism

EC Number Organism UniProt Comment Textmining
5.4.3.3 Fusobacterium nucleatum Q8RHX7 AND Q8RHX8 alpha- and beta-subunits
-
5.4.3.5 Acetoanaerobium sticklandii E3PY95 AND E3PY96 subunits beta and alpha
-
5.4.3.5 Acetoanaerobium sticklandii DSM 519 E3PY95 AND E3PY96 subunits beta and alpha
-

Reaction

EC Number Reaction Comment Organism Reaction ID
5.4.3.5 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 Acetoanaerobium sticklandii
a

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
5.4.3.3 (3S)-3,6-diaminohexanoate
-
 Fusobacterium nucleatum (3S,5S)-3,5-diaminohexanoate
-
 r
5.4.3.3 D-lysine
-
 Fusobacterium nucleatum 2,5-diaminohexanoate
-
 r
5.4.3.3 additional information lysine 5,6-aminomutase (5,6-LAM) can perform 1,2-amino shifts on L-beta-lysine as well as D- and L-lysine. Docking of L-lysine or L-beta-lysine into the active site of 5,6-LAM revealed distinct non-covalent interactions between the substrate and the enzyme compared to that of the ornithine 4,5-aminomutase (EC 5.4.3.5) substrate complex. In 5,6-LAM, K370alpha forms a salt bridge with the alpha-carboxylate of the lysyl-pyridoxal 5'-phosphate complex, while the alpha-amine interacts with D298alpha. These two substitutions presumably increase the binding cavity of 5,6-LAM to accommodate different isomers of lysine. 5,6-LAM is able to act on D-lysine Fusobacterium nucleatum ?
-
 ?
5.4.3.5 D-ornithine
-
 Acetoanaerobium sticklandii (2R,4S)-2,4-diaminopentanoate
-
 r
5.4.3.5 D-ornithine
-
 Acetoanaerobium sticklandii DSM 519 (2R,4S)-2,4-diaminopentanoate
-
 r
5.4.3.5 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 Acetoanaerobium sticklandii ?
-
 ?
5.4.3.5 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 Acetoanaerobium sticklandii DSM 519 ?
-
 ?

Subunits

EC Number Subunits Comment Organism
5.4.3.3 heterotetramer alpha2beta2 Fusobacterium nucleatum
5.4.3.5 heterotetramer alpha2beta2 Acetoanaerobium sticklandii

Synonyms

EC Number Synonyms Comment Organism
5.4.3.3 LAM
-
 Fusobacterium nucleatum
5.4.3.5 OAM
-
 Acetoanaerobium sticklandii
5.4.3.5 ornithine 4,5-aminomutase
-
 Acetoanaerobium sticklandii

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
5.4.3.3 25
-
 assay at Fusobacterium nucleatum
5.4.3.5 25
-
 assay at Acetoanaerobium sticklandii

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
5.4.3.5 0.002
-
 D-ornithine pH 8.5, 25°C, recombinant beta-subunit mutant E81D Acetoanaerobium sticklandii
5.4.3.5 0.013
-
 D-ornithine pH 8.5, 25°C, recombinant beta-subunit mutant E81A Acetoanaerobium sticklandii
5.4.3.5 0.027
-
 D-ornithine pH 8.5, 25°C, recombinant beta-subunit mutant Y160F Acetoanaerobium sticklandii
5.4.3.5 0.029
-
 D-ornithine pH 8.5, 25°C, recombinant beta-subunit mutant E81Q Acetoanaerobium sticklandii
5.4.3.5 0.068
-
 D-ornithine pH 8.5, 25°C, recombinant beta-subunit mutant R297K Acetoanaerobium sticklandii
5.4.3.5 0.12
-
 D-ornithine pH 8.5, 25°C, recombinant beta-subunit mutant Y187F Acetoanaerobium sticklandii
5.4.3.5 0.37
-
 D-ornithine pH 8.5, 25°C, recombinant beta-subunit mutant S162A Acetoanaerobium sticklandii
5.4.3.5 0.38
-
 D-ornithine pH 8.5, 25°C, recombinant beta-subunit mutant N226D Acetoanaerobium sticklandii
5.4.3.5 8.2
-
 D-ornithine pH 8.5, 25°C, recombinant wild-type enzyme Acetoanaerobium sticklandii

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
5.4.3.3 8.5
-
 assay at Fusobacterium nucleatum
5.4.3.5 8.5
-
 assay at Acetoanaerobium sticklandii

Cofactor

EC Number Cofactor Comment Organism Structure
5.4.3.3 5'-deoxyadenosylcobalamin
-
 Fusobacterium nucleatum
5.4.3.3 additional information lysine 5,6-aminomutase is a 5'-deoxyadenosylcobalamin and pyridoxal 5'-phosphate co-dependent radical enzyme Fusobacterium nucleatum
5.4.3.3 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 ornithine 4,5-aminomutase, EC 5.4.3.5 Fusobacterium nucleatum
5.4.3.5 5'-deoxyadenosylcobalamin
-
 Acetoanaerobium sticklandii
5.4.3.5 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 Acetoanaerobium sticklandii
5.4.3.5 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 Acetoanaerobium sticklandii

Ki Value [mM]

EC Number Ki Value [mM] Ki Value maximum [mM] Inhibitor Comment Organism Structure
5.4.3.5 0.0046
-
 2,4-diaminobutyrate pH 8.5, 25°C, recombinant wild-type enzyme Acetoanaerobium sticklandii
5.4.3.5 0.032
-
 2,4-diaminobutyrate pH 8.5, 25°C, recombinant mutant Y187F Acetoanaerobium sticklandii
5.4.3.5 0.1
-
 2,4-diaminobutyrate pH 8.5, 25°C, recombinant mutant N226D Acetoanaerobium sticklandii
5.4.3.5 0.27
-
 2,4-diaminobutyrate pH 8.5, 25°C, recombinant mutant S162A Acetoanaerobium sticklandii
5.4.3.5 2
-
 2,4-diaminobutyrate pH 8.5, 25°C, recombinant mutant Y160F Acetoanaerobium sticklandii

General Information

EC Number General Information Comment Organism
5.4.3.3 additional information lysine 5,6-aminomutase (5,6-LAM) from Clostridium sticklandii is an adenosylcobalamin (AdoCbl) and pyridoxal 5'-phosphate (PLP)-dependent enzyme Fusobacterium nucleatum
5.4.3.5 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 Acetoanaerobium sticklandii
5.4.3.5 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 Acetoanaerobium sticklandii

kcat/KM [mM/s]

EC Number kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
5.4.3.5 0.014
-
 D-ornithine pH 8.5, 25°C, recombinant beta-subunit mutant R297K Acetoanaerobium sticklandii
5.4.3.5 0.016
-
 D-ornithine pH 8.5, 25°C, recombinant beta-subunit mutant E81A Acetoanaerobium sticklandii
5.4.3.5 0.19
-
 D-ornithine pH 8.5, 25°C, recombinant beta-subunit mutant Y160F Acetoanaerobium sticklandii
5.4.3.5 0.33
-
 D-ornithine pH 8.5, 25°C, recombinant beta-subunit mutant E81Q Acetoanaerobium sticklandii
5.4.3.5 3.2
-
 D-ornithine pH 8.5, 25°C, recombinant beta-subunit mutant N226D Acetoanaerobium sticklandii
5.4.3.5 3.9
-
 D-ornithine pH 8.5, 25°C, recombinant beta-subunit mutant Y187F Acetoanaerobium sticklandii
5.4.3.5 13
-
 D-ornithine pH 8.5, 25°C, recombinant beta-subunit mutant S162A Acetoanaerobium sticklandii
5.4.3.5 190
-
 D-ornithine pH 8.5, 25°C, recombinant wild-type enzyme Acetoanaerobium sticklandii