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Literature summary for 1.21.3.3 extracted from

  • Gaweska, H.M.; Roberts, K.M.; Fitzpatrick, P.F.
    Isotope effects suggest a stepwise mechanism for berberine bridge enzyme (2012), Biochemistry, 51, 7342-7347.
    View publication on PubMedView publication on EuropePMC

Cloned(Commentary)

Cloned (Comment) Organism
expression in Pichia pastoris Eschscholzia californica

Protein Variants

Protein Variants Comment Organism
E417Q solvent isotope effects on kred are equal to 1 for both wild-type and the E417Q mutant, indicating that solvent exchangeable protons are not in flight during or before flavin reduction, thus eliminating a fully concerted mechanism Eschscholzia californica

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
0.00027
-
(S)-reticuline wild-type, 25°C, pH 9.0 Eschscholzia californica
0.28
-
O2 wild-type, 25°C, pH 9.0 Eschscholzia californica

Organism

Organism UniProt Comment Textmining
Eschscholzia californica
-
-
-

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
(S)-coreximine + O2
-
Eschscholzia californica (13aS)-2,11-dihydroxy-3,10-dimethoxy-5,8,13,13a-tetrahydroisoquinolino[3,2-a]isoquinolin-7-ium + H2O2
-
?
(S)-laudanosine + H2O2
-
Eschscholzia californica ? + O2
-
?
(S)-N-methylcoclaurine + O2
-
Eschscholzia californica (S)-coclaurine + H2O2
-
?
(S)-norsteponine + H2O2
-
Eschscholzia californica ? + O2
-
?
(S)-reticuline + O2
-
Eschscholzia californica (S)-scoulerine + H2O2
-
r
(S)-scoulerine + H2O2
-
Eschscholzia californica (S)-reticuline + O2
-
r
6,7-dimethoxy-2-methyl-1,2,3,4-tetrahydroisoquinoline + H2O2
-
Eschscholzia californica ? + O2
-
?
additional information C-H bond cleavage is rate-limiting during flavin reduction. Solvent isotope effects on kred indicate that solvent exchangeable protons are not in flight during or before flavin reduction, thus eliminating a fully concerted mechanism. Deprotonation is not occurring before or during C-H bond cleavage, and a hydroxyl group must be present at C3 for cyclization. This could be due to the methoxy group being less electron-donating than a hydroxyl substituent or to disruption of the interaction between the glutamate and the substrate, causing altered binding. A concerted attack of a methyl amine by the C2-atom and hydride transfer is less likely than attack on a methylene iminium ion intermediate by the C2-atom, indicating that a stepwise mechanism is the likely mechanism Eschscholzia californica ?
-
?

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
5.4
-
(S)-reticuline wild-type, 25°C, pH 9.0 Eschscholzia californica
10.5
-
O2 wild-type, 25°C, pH 9.0 Eschscholzia californica

kcat/KM [mM/s]

kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
37
-
O2 wild-type, 25°C, pH 9.0 Eschscholzia californica
20000
-
(S)-reticuline wild-type, 25°C, pH 9.0 Eschscholzia californica