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Literature summary for 2.3.1.220 extracted from

  • Klundt, T.; Bocola, M.; Luetge, M.; Beuerle, T.; Liu, B.; Beerhues, L.
    A single amino acid substitution converts benzophenone synthase into phenylpyrone synthase (2009), J. Biol. Chem., 284, 30957-30964.
    View publication on PubMedView publication on EuropePMC
Search for more literature for 2.3.1.220

Cloned(Commentary)

Cloned (Comment) Organism
expression of N-terminally His-tagged wild-type and mutant enzymes in Escherichia coli Hypericum androsaemum

Protein Variants

Protein Variants Comment Organism
T135L the T135L mutant catalyzes the addition of only two acetyl groups to the benzoyl starter unit. The triketide is the final linear intermediate and cyclizes into phenylpyrone via C-5 keto-enol oxygen -> C-1 lactonization. The T135L substitution opens a new pocket, the entrance of which is blocked in the wild-type enzyme by hydrogen bond formation between the threonine side chain and the backbone. Because of the interaction of the lipophilic side chain of the introduced leucine residue with the phenyl group of the growing polyketide chain, the triketide in the active site cavity of the T135L mutant may be redirected into the new pocket Hypericum androsaemum

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
0.001
-
 benzoyl-CoA pH 7.0, 35°C, mutant T135L enzyme Hypericum androsaemum
0.0086
-
 benzoyl-CoA pH 7.0, 35°C, wild-type enzyme Hypericum androsaemum
0.0087
-
 malonyl-CoA pH 7.0, 35°C, mutant T135L enzyme Hypericum androsaemum
0.0313
-
 malonyl-CoA pH 7.0, 35°C, wild-type enzyme Hypericum androsaemum

Molecular Weight [Da]

Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
42800
-
 2 * 42800 Hypericum androsaemum
85000
-
-
 Hypericum androsaemum

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
3 malonyl-CoA + benzoyl-CoA Hypericum androsaemum
-
 4 CoA + 2,4,6-trihydroxybenzophenone + 3 CO2
-
 ?

Organism

Organism UniProt Comment Textmining
Hypericum androsaemum
-
-
-

Purification (Commentary)

Purification (Comment) Organism
reccombinant N-terminally His-tagged wild-type and mutant enzymes from Escherichia coli by nickel affinity chromatography Hypericum androsaemum

Reaction

Reaction Comment Organism Reaction ID
3 malonyl-CoA + benzoyl-CoA = 4 CoA + 2,4,6-trihydroxybenzophenone + 3 CO2 the enzyme catalyzes the iterative condensation of benzoyl-CoA with three molecules of malonyl-CoA to give a linear tetraketide intermediate, which is subsequently cyclized into 2,4,6-trihydroxybenzophenone via intramolecular Claisen condensation, catalytic mechanism, overview Hypericum androsaemum
a

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
2 malonyl-CoA + benzoyl-CoA reaction of mutant T135L, no activity with the wild-type enzyme. The T135L mutant adds only two acetyl groups to the benzoyl starter unit to form a triketide intermediate which then cyclized into 6-phenyl-4-hydroxy-2-pyrone via C5 keto-enol oxygen -> C1 lactonization Hypericum androsaemum 3 CoA + 6-phenyl-4-hydroxy-2-pyrone + 2 CO2
-
 ?
3 malonyl-CoA + benzoyl-CoA
-
 Hypericum androsaemum 4 CoA + 2,4,6-trihydroxybenzophenone + 3 CO2
-
 ?
3 malonyl-CoA + benzoyl-CoA benzoyl-CoA is the preferred starter substrate of the wild-type enzyme, activity by wild-type enzyme and mutant T135L Hypericum androsaemum 4 CoA + 2,4,6-trihydroxybenzophenone + 3 CO2
-
 ?
additional information 3-hydroxybenzoyl-CoA is the second best starter substrate for the wild-type enzyme but a poor starter molecule for the mutant enzyme T135L, resulting in formation of 2,3',4,6-tetrahydroxybenzophenone, reaction of EC 2.3.1.151. Product identification by mass spectrometry. No activity by wild-type enzyme and mutant T135L with 2-hydroxybenzoyl-CoA, 4-hydroxybenzoyl-CoA, cinnamoyl-CoA, 2-coumaroyl-CoA, 3-coumaroyl-CoA, 4-coumaroyl-CoA, and acetyl-CoA Hypericum androsaemum ?
-
 ?

Subunits

Subunits Comment Organism
homodimer 2 * 42800 Hypericum androsaemum

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
35
-
-
 Hypericum androsaemum

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
0.0068
-
 malonyl-CoA pH 7.0, 35°C, mutant T135L enzyme Hypericum androsaemum
0.0068
-
 benzoyl-CoA pH 7.0, 35°C, mutant T135L enzyme Hypericum androsaemum
0.055
-
 malonyl-CoA pH 7.0, 35°C, wild-type enzyme Hypericum androsaemum
0.055
-
 benzoyl-CoA pH 7.0, 35°C, wild-type enzyme Hypericum androsaemum

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
6.5 7
-
 Hypericum androsaemum

pI Value

Organism Comment pI Value Maximum pI Value
Hypericum androsaemum
-
-
 5.78

General Information

General Information Comment Organism
evolution BPS is a type III polyketide synthase, PKS, and part of the chalcone synthase group of the superfamily of enzymes Hypericum androsaemum
malfunction the T135L mutant catalyzes the addition of only two acetyl groups to the benzoyl starter unit. The triketide is the final linear intermediate and cyclizes into phenylpyrone via C-5 keto-enol oxygen -> C-1 lactonization Hypericum androsaemum
additional information residues involved in the initiation pocket are M217, I258, A260, and Y269, and in the elongation pocket T135, S136, T197, M199, T200, S219, M267, and G342, the catalytic triad is formed by residues C167, H307, and N340, molecular modeling constructed based on the crystal structure of Medicago sativa CHS2 complexed with resveratrol, PDB ID 1CGZ, overview Hypericum androsaemum

kcat/KM [mM/s]

kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
0.0064
-
 malonyl-CoA pH 7.0, 35°C, wild-type enzyme Hypericum androsaemum
0.0064
-
 benzoyl-CoA pH 7.0, 35°C, wild-type enzyme Hypericum androsaemum
0.0068
-
 malonyl-CoA pH 7.0, 35°C, mutant T135L enzyme Hypericum androsaemum
0.0068
-
 benzoyl-CoA pH 7.0, 35°C, mutant T135L enzyme Hypericum androsaemum