Reaction #470402

ord-d79a748661f74249bb00c9cbd97ff172

Reaction equation

CC(C)[C@H](N)C(=O)O
L-valine
N[C@@H](CCC(=O)O)C(=O)O
L-glutamic acid
O=C(O)CCC(=O)C(=O)O
2-oxoglutaric acid
CC[C@H](C)[C@H](N)C(=O)O
L-isoleucine
O=S(=O)(O)CCN1CCN(CCO)CC1
HEPES
O=C1O[C@H]([C@@H](O)CO)C(O)=C1O
ascorbic acid
NCCCC[C@H](N)C(=O)O
L-lysine
→
CC(O)[C@H](C)[C@H](N)C(=O)O
4-hydroxyisoleucine

Reactants

CC(C)[C@H](N)C(=O)O
L-valine
—
2-e
—
amino acids
N[C@@H](CCC(=O)O)C(=O)O
L-glutamic acid
—
amino acid
—
amino acid
O=C(O)CCC(=O)C(=O)O
2-oxoglutaric acid
—
Fe2+
CC[C@H](C)[C@H](N)C(=O)O
L-isoleucine
—
amino acids
O=S(=O)(O)CCN1CCN(CCO)CC1
HEPES
O=C1O[C@H]([C@@H](O)CO)C(O)=C1O
ascorbic acid
NCCCC[C@H](N)C(=O)O
L-lysine

Reagents

None

Conditions

Detailed conditions
See reaction.notes.procedure_details.

Workup

  1. 1
    workup.ADDITIONThe cell lysate and a substrate solution were mixed
  2. 2
    OtherThe substrate reaction mixture

Procedure

By using a cell lysate prepared from 2-e-2 cells at OD660 of 7 according to the method described in <5>, the reaction characteristics for various amino acids were evaluated. The cell lysate and a substrate solution were mixed, then the reaction proceeded at 30° C. for 1 hour, and the production of new substances was evaluated by TLC or amino acid analysis. The substrate reaction mixture contained 5 mM amino acid, 5 mM Fe2+, 5 mM 2-oxoglutaric acid, 5 mM ascorbic acid, and 100 mM HEPES (pH 7). In addition to L-isoleucine, the amino acids L-leucine, L-valine, L-glutamic acid, and L-lysine were each individually evaluated. The 4-hydroxyisoleucine which was produced was analyzed by the method described in Example 1. The results are shown in Table 6. Production of amino acids other than L-isoleucine was not observed. Therefore, it was suggested that this enzyme was an isoleucine-specific dioxygenase.

Source

DOI: 10.6084/m9.figshare.5104873.v1Patent: US08367381B2uspto-grants-2013_02