Cloned (Comment) | Organism |
---|---|
genes oraS and oraE encode the alpha- and beta-subunit, respectively | Acetoanaerobium sticklandii |
Protein Variants | Comment | Organism |
---|---|---|
E81A | site-directed mutagenesis, inactive beta-subunit mutant | Acetoanaerobium sticklandii |
E81D | site-directed mutagenesis, almost inactive beta-subunit mutant | Acetoanaerobium sticklandii |
E81Q | site-directed mutagenesis, the beta-subunit mutant shows highly reduced activity compared to wild-type | Acetoanaerobium sticklandii |
N226D | site-directed mutagenesis, the beta-subunit mutant shows highly reduced activity compared to wild-type | Acetoanaerobium sticklandii |
R297K | site-directed mutagenesis, almost inactive beta-subunit mutant | Acetoanaerobium sticklandii |
S162A | site-directed mutagenesis, the beta-subunit mutant shows highly reduced activity compared to wild-type | Acetoanaerobium sticklandii |
Y160F | site-directed mutagenesis, almost inactive beta-subunit mutant | Acetoanaerobium sticklandii |
Y187F | site-directed mutagenesis, the beta-subunit mutant shows highly reduced activity compared to wild-type | Acetoanaerobium sticklandii |
Inhibitors | Comment | Organism | Structure |
---|---|---|---|
2,4-diaminobutyrate | - |
Acetoanaerobium sticklandii |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.029 | - |
D-ornithine | pH 8.5, 25°C, recombinant beta-subunit mutant S162A | Acetoanaerobium sticklandii | |
0.031 | - |
D-ornithine | pH 8.5, 25°C, recombinant beta-subunit mutant Y187F | Acetoanaerobium sticklandii | |
0.043 | - |
D-ornithine | pH 8.5, 25°C, recombinant wild-type enzyme | Acetoanaerobium sticklandii | |
0.12 | - |
D-ornithine | pH 8.5, 25°C, recombinant beta-subunit mutant N226D | Acetoanaerobium sticklandii | |
0.14 | - |
D-ornithine | pH 8.5, 25°C, recombinant beta-subunit mutant Y160F | Acetoanaerobium sticklandii | |
0.78 | - |
D-ornithine | pH 8.5, 25°C, recombinant beta-subunit mutant E81A | Acetoanaerobium sticklandii | |
0.87 | - |
D-ornithine | pH 8.5, 25°C, recombinant beta-subunit mutant E81Q | Acetoanaerobium sticklandii | |
4.9 | - |
D-ornithine | pH 8.5, 25°C, recombinant beta-subunit mutant R297K | Acetoanaerobium sticklandii |
Molecular Weight [Da] | Molecular Weight Maximum [Da] | Comment | Organism |
---|---|---|---|
191400 | - |
- |
Acetoanaerobium sticklandii |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
D-ornithine | Acetoanaerobium sticklandii | - |
(2R,4S)-2,4-diaminopentanoate | - |
r | |
D-ornithine | Acetoanaerobium sticklandii DSM 519 | - |
(2R,4S)-2,4-diaminopentanoate | - |
r |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Acetoanaerobium sticklandii | E3PY95 AND E3PY96 | subunits beta and alpha | - |
Acetoanaerobium sticklandii DSM 519 | E3PY95 AND E3PY96 | subunits beta and alpha | - |
Reaction | Comment | Organism | Reaction ID |
---|---|---|---|
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 |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
D-ornithine | - |
Acetoanaerobium sticklandii | (2R,4S)-2,4-diaminopentanoate | - |
r | |
D-ornithine | - |
Acetoanaerobium sticklandii DSM 519 | (2R,4S)-2,4-diaminopentanoate | - |
r | |
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 | ? | - |
? | |
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 | Comment | Organism |
---|---|---|
heterotetramer | alpha2beta2 | Acetoanaerobium sticklandii |
Synonyms | Comment | Organism |
---|---|---|
OAM | - |
Acetoanaerobium sticklandii |
ornithine 4,5-aminomutase | - |
Acetoanaerobium sticklandii |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
25 | - |
assay at | Acetoanaerobium sticklandii |
Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.002 | - |
D-ornithine | pH 8.5, 25°C, recombinant beta-subunit mutant E81D | Acetoanaerobium sticklandii | |
0.013 | - |
D-ornithine | pH 8.5, 25°C, recombinant beta-subunit mutant E81A | Acetoanaerobium sticklandii | |
0.027 | - |
D-ornithine | pH 8.5, 25°C, recombinant beta-subunit mutant Y160F | Acetoanaerobium sticklandii | |
0.029 | - |
D-ornithine | pH 8.5, 25°C, recombinant beta-subunit mutant E81Q | Acetoanaerobium sticklandii | |
0.068 | - |
D-ornithine | pH 8.5, 25°C, recombinant beta-subunit mutant R297K | Acetoanaerobium sticklandii | |
0.12 | - |
D-ornithine | pH 8.5, 25°C, recombinant beta-subunit mutant Y187F | Acetoanaerobium sticklandii | |
0.37 | - |
D-ornithine | pH 8.5, 25°C, recombinant beta-subunit mutant S162A | Acetoanaerobium sticklandii | |
0.38 | - |
D-ornithine | pH 8.5, 25°C, recombinant beta-subunit mutant N226D | Acetoanaerobium sticklandii | |
8.2 | - |
D-ornithine | pH 8.5, 25°C, recombinant wild-type enzyme | Acetoanaerobium sticklandii |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
8.5 | - |
assay at | Acetoanaerobium sticklandii |
Cofactor | Comment | Organism | Structure |
---|---|---|---|
5'-deoxyadenosylcobalamin | - |
Acetoanaerobium sticklandii | |
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 | |
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] | Ki Value maximum [mM] | Inhibitor | Comment | Organism | Structure |
---|---|---|---|---|---|
0.0046 | - |
2,4-diaminobutyrate | pH 8.5, 25°C, recombinant wild-type enzyme | Acetoanaerobium sticklandii | |
0.032 | - |
2,4-diaminobutyrate | pH 8.5, 25°C, recombinant mutant Y187F | Acetoanaerobium sticklandii | |
0.1 | - |
2,4-diaminobutyrate | pH 8.5, 25°C, recombinant mutant N226D | Acetoanaerobium sticklandii | |
0.27 | - |
2,4-diaminobutyrate | pH 8.5, 25°C, recombinant mutant S162A | Acetoanaerobium sticklandii | |
2 | - |
2,4-diaminobutyrate | pH 8.5, 25°C, recombinant mutant Y160F | Acetoanaerobium sticklandii |
General Information | Comment | Organism |
---|---|---|
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 |
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 Value [1/mMs-1] | kcat/KM Value Maximum [1/mMs-1] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.014 | - |
D-ornithine | pH 8.5, 25°C, recombinant beta-subunit mutant R297K | Acetoanaerobium sticklandii | |
0.016 | - |
D-ornithine | pH 8.5, 25°C, recombinant beta-subunit mutant E81A | Acetoanaerobium sticklandii | |
0.19 | - |
D-ornithine | pH 8.5, 25°C, recombinant beta-subunit mutant Y160F | Acetoanaerobium sticklandii | |
0.33 | - |
D-ornithine | pH 8.5, 25°C, recombinant beta-subunit mutant E81Q | Acetoanaerobium sticklandii | |
3.2 | - |
D-ornithine | pH 8.5, 25°C, recombinant beta-subunit mutant N226D | Acetoanaerobium sticklandii | |
3.9 | - |
D-ornithine | pH 8.5, 25°C, recombinant beta-subunit mutant Y187F | Acetoanaerobium sticklandii | |
13 | - |
D-ornithine | pH 8.5, 25°C, recombinant beta-subunit mutant S162A | Acetoanaerobium sticklandii | |
190 | - |
D-ornithine | pH 8.5, 25°C, recombinant wild-type enzyme | Acetoanaerobium sticklandii |