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Literature summary extracted from

  • 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 (2014), Biochemistry, 53, 5432-5443 .
    View publication on PubMed

Cloned(Commentary)

EC Number Cloned (Comment) Organism
5.4.3.5 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 (Commentary)

EC Number Crystallization (Comment) Organism
5.4.3.5 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

EC Number Protein Variants Comment Organism
5.4.3.5 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
5.4.3.5 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]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
5.4.3.5 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
5.4.3.5 0.068
-
 DL-Ornithine pH 8.5, 20°C, recombinant beta-subunit mutant Y187A Acetoanaerobium sticklandii
5.4.3.5 0.162
-
 DL-Ornithine pH 8.5, 20°C, recombinant beta-subunit mutant Y187F Acetoanaerobium sticklandii
5.4.3.5 0.567
-
 DL-Ornithine pH 8.5, 20°C, recombinant wild-type enzyme Acetoanaerobium sticklandii

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
5.4.3.5 D-ornithine Acetoanaerobium sticklandii
-
 (2R,4S)-2,4-diaminopentanoate
-
 r

Organism

EC Number Organism UniProt Comment Textmining
5.4.3.5 Acetoanaerobium sticklandii E3PY95 AND E3PY96 subunits beta and alpha
-

Purification (Commentary)

EC Number Purification (Comment) Organism
5.4.3.5 recombinant His-tagged wild-type and beta-subunit mutant enzymes from Escherichia coli Acetoanaerobium sticklandii

Reaction

EC Number Reaction Comment Organism Reaction ID
5.4.3.5 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
a

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
5.4.3.5 D-ornithine
-
 Acetoanaerobium sticklandii (2R,4S)-2,4-diaminopentanoate
-
 r
5.4.3.5 DL-ornithine
-
 Acetoanaerobium sticklandii (2R,4S)-2,4-diaminopentanoate
-
 r
5.4.3.5 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

EC Number Subunits Comment Organism
5.4.3.5 heterotetramer alpha2beta2, 2 * 12800, alpha-subunit, + 2 * 82900, beta-subunit, SDS-PAGE Acetoanaerobium sticklandii
5.4.3.5 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

EC Number Synonyms Comment Organism
5.4.3.5 adenosylcobalamin-dependent ornithine 4,5-aminomutase
-
 Acetoanaerobium sticklandii
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
5.4.3.5 20
-
 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 0.0023
-
 DL-Ornithine pH 8.5, 20°C, recombinant beta-subunit mutant Y187A Acetoanaerobium sticklandii
5.4.3.5 0.115
-
 DL-Ornithine pH 8.5, 20°C, recombinant beta-subunit mutant Y187F Acetoanaerobium sticklandii
5.4.3.5 2.9
-
 DL-Ornithine pH 8.5, 20°C, recombinant wild-type enzyme Acetoanaerobium sticklandii

pH Optimum

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

Cofactor

EC Number Cofactor Comment Organism Structure
5.4.3.5 adenosylcobalamin dependent on Acetoanaerobium sticklandii
5.4.3.5 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
5.4.3.5 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

EC Number General Information Comment Organism
5.4.3.5 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 [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 0.034
-
 DL-Ornithine pH 8.5, 20°C, recombinant beta-subunit mutant Y187A Acetoanaerobium sticklandii
5.4.3.5 0.71
-
 DL-Ornithine pH 8.5, 20°C, recombinant beta-subunit mutant Y187F Acetoanaerobium sticklandii
5.4.3.5 5.11
-
 DL-Ornithine pH 8.5, 20°C, recombinant wild-type enzyme Acetoanaerobium sticklandii