Cloned (Comment) | Organism |
---|---|
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 | Acetoanaerobium sticklandii |
Crystallization (Comment) | Organism |
---|---|
analysis of the crystal structure of the active site of ornithine 4,5-aminomutase complexed with the natural substrate D-ornithine, PDB ID 3KOZ | Acetoanaerobium sticklandii |
Protein Variants | Comment | Organism |
---|---|---|
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 | Acetoanaerobium sticklandii |
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 | Acetoanaerobium sticklandii |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
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 | Acetoanaerobium sticklandii | |
0.068 | - |
DL-Ornithine | pH 8.5, 20°C, recombinant beta-subunit mutant Y187A | Acetoanaerobium sticklandii | |
0.162 | - |
DL-Ornithine | pH 8.5, 20°C, recombinant beta-subunit mutant Y187F | Acetoanaerobium sticklandii | |
0.567 | - |
DL-Ornithine | pH 8.5, 20°C, recombinant wild-type enzyme | 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 |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Acetoanaerobium sticklandii | E3PY95 AND E3PY96 | subunits beta and alpha | - |
Purification (Comment) | Organism |
---|---|
recombinant His-tagged wild-type and beta-subunit mutant enzymes from Escherichia coli | Acetoanaerobium sticklandii |
Reaction | Comment | Organism | Reaction ID |
---|---|---|---|
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 | Acetoanaerobium sticklandii |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
D-ornithine | - |
Acetoanaerobium sticklandii | (2R,4S)-2,4-diaminopentanoate | - |
r | |
DL-ornithine | - |
Acetoanaerobium sticklandii | (2R,4S)-2,4-diaminopentanoate | - |
r | |
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 | Acetoanaerobium sticklandii | ? | - |
? |
Subunits | Comment | Organism |
---|---|---|
heterotetramer | alpha2beta2, 2 * 12800, alpha-subunit, + 2 * 82900, beta-subunit, SDS-PAGE | Acetoanaerobium sticklandii |
More | 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 | Acetoanaerobium sticklandii |
Synonyms | Comment | Organism |
---|---|---|
adenosylcobalamin-dependent ornithine 4,5-aminomutase | - |
Acetoanaerobium sticklandii |
OAM | - |
Acetoanaerobium sticklandii |
ornithine 4,5-aminomutase | - |
Acetoanaerobium sticklandii |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
20 | - |
assay at | Acetoanaerobium sticklandii |
Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.0023 | - |
DL-Ornithine | pH 8.5, 20°C, recombinant beta-subunit mutant Y187A | Acetoanaerobium sticklandii | |
0.115 | - |
DL-Ornithine | pH 8.5, 20°C, recombinant beta-subunit mutant Y187F | Acetoanaerobium sticklandii | |
2.9 | - |
DL-Ornithine | pH 8.5, 20°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 |
---|---|---|---|
adenosylcobalamin | dependent on | Acetoanaerobium sticklandii | |
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 | Acetoanaerobium sticklandii | |
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 | Acetoanaerobium sticklandii |
General Information | Comment | Organism |
---|---|---|
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 | Acetoanaerobium sticklandii |
kcat/KM Value [1/mMs-1] | kcat/KM Value Maximum [1/mMs-1] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.034 | - |
DL-Ornithine | pH 8.5, 20°C, recombinant beta-subunit mutant Y187A | Acetoanaerobium sticklandii | |
0.71 | - |
DL-Ornithine | pH 8.5, 20°C, recombinant beta-subunit mutant Y187F | Acetoanaerobium sticklandii | |
5.11 | - |
DL-Ornithine | pH 8.5, 20°C, recombinant wild-type enzyme | Acetoanaerobium sticklandii |