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Literature summary 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

Cloned(Commentary)

EC Number Cloned (Comment) Organism
2.3.1.151 wild-type benzophenone synthase and enzyme mutants are heterologously expressed as N-terminally His6-tagged proteins in Escherichia coli Hypericum androsaemum
2.3.1.220 expression of N-terminally His-tagged wild-type and mutant enzymes in Escherichia coli Hypericum androsaemum

Protein Variants

EC Number Protein Variants Comment Organism
2.3.1.151 T135A inactive enzyme Hypericum androsaemum
2.3.1.151 T135F mutant functionally resemble the wild-type enzyme, albeit with reduced catalytic activities Hypericum androsaemum
2.3.1.151 T135G inactive enzyme Hypericum androsaemum
2.3.1.151 T135I inactive enzyme Hypericum androsaemum
2.3.1.151 T135L a point mutation in the active site cavity transforms benzophenone synthase into a functional phenylpyrone synthase (PPS). The dramatic change in both substrate and product specificities of benzophenone synthase is rationalized by homology modeling. The mutation may open a new pocket that accommodates the phenyl moiety of the triketide intermediate but limits polyketide elongation to two reactions, resulting in phenylpyrone formation. kcat (substrate: benzoyl-CoA): 0.0086/sec, Km (benzoyl-CoA): 0.001 mM, Km (malonyl-CoA): 8.7 mM. In contrast to the wild-type enzyme, the T135L mutant forms phenylpyrone as a major product and only traces of 2,4,6-trihydroxybenzophenone when incubated with benzoyl-CoA and malonyl-CoA. T135L mutant is almost inactive with 3-hydroxybenzoyl-CoA Hypericum androsaemum
2.3.1.151 T135N inactive enzyme Hypericum androsaemum
2.3.1.151 T135S mutant functionally resemble the wild-type enzyme, albeit with reduced catalytic activities Hypericum androsaemum
2.3.1.151 T135V inactive enzyme Hypericum androsaemum
2.3.1.151 T135Y inactive enzyme Hypericum androsaemum
2.3.1.220 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]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
2.3.1.151 0.001
-
 benzoyl-CoA mutant T135L Hypericum androsaemum
2.3.1.151 0.0086
-
 benzoyl-CoA wild-type Hypericum androsaemum
2.3.1.151 0.0087
-
 malonyl-CoA mutant T135L Hypericum androsaemum
2.3.1.151 0.031
-
 malonyl-CoA wild-type Hypericum androsaemum
2.3.1.220 0.001
-
 benzoyl-CoA pH 7.0, 35°C, mutant T135L enzyme Hypericum androsaemum
2.3.1.220 0.0086
-
 benzoyl-CoA pH 7.0, 35°C, wild-type enzyme Hypericum androsaemum
2.3.1.220 0.0087
-
 malonyl-CoA pH 7.0, 35°C, mutant T135L enzyme Hypericum androsaemum
2.3.1.220 0.0313
-
 malonyl-CoA pH 7.0, 35°C, wild-type enzyme Hypericum androsaemum

Molecular Weight [Da]

EC Number Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
2.3.1.151 43000
-
 SDS-PAGE, molecular weight of mutant T135L Hypericum androsaemum
2.3.1.220 42800
-
 2 * 42800 Hypericum androsaemum
2.3.1.220 85000
-
-
 Hypericum androsaemum

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
2.3.1.220 3 malonyl-CoA + benzoyl-CoA Hypericum androsaemum
-
 4 CoA + 2,4,6-trihydroxybenzophenone + 3 CO2
-
 ?

Organism

EC Number Organism UniProt Comment Textmining
2.3.1.151 Hypericum androsaemum
-
-
-
2.3.1.220 Hypericum androsaemum
-
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
2.3.1.151 purified on a nickel-nitrilotriacetic acid affinity matrix Hypericum androsaemum
2.3.1.220 reccombinant N-terminally His-tagged wild-type and mutant enzymes from Escherichia coli by nickel affinity chromatography Hypericum androsaemum

Reaction

EC Number Reaction Comment Organism Reaction ID
2.3.1.220 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)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
2.3.1.151 2 malonyl-CoA + benzoyl-CoA relative activity: 100%, wildtype benzophenone synthase forms 2,4,6-trihydroxybenzophenone as a major product when incubated with benzoyl-CoA and malonyl-CoA, along with small amounts of 6-phenyl-4-hydroxy-2-pyrone Hypericum androsaemum 3 CoA + 2,4,6-trihydroxybenzophenone + 2 CO2
-
 ?
2.3.1.151 3 malonyl-CoA + 3-hydroxybenzoyl-CoA relative activity: 57% Hypericum androsaemum 4 CoA + 2,3',4,6-tetrahydroxybenzophenone + 3 CO2
-
 ?
2.3.1.151 3 malonyl-CoA + benzoyl-CoA relative activity: 100%, wildtype benzophenone synthase forms 2,4,6-trihydroxybenzophenone as a major product when incubated with benzoyl-CoA and malonyl-CoA, along with small amounts of 6-phenyl-4-hydroxy-2-pyrone Hypericum androsaemum 4 CoA + 6-phenyl-4-hydroxy-2-pyrone + 3 CO2
-
 ?
2.3.1.220 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
-
 ?
2.3.1.220 3 malonyl-CoA + benzoyl-CoA
-
 Hypericum androsaemum 4 CoA + 2,4,6-trihydroxybenzophenone + 3 CO2
-
 ?
2.3.1.220 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
-
 ?
2.3.1.220 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

EC Number Subunits Comment Organism
2.3.1.220 homodimer 2 * 42800 Hypericum androsaemum

Synonyms

EC Number Synonyms Comment Organism
2.3.1.151 benzophenone synthase
-
 Hypericum androsaemum
2.3.1.151 BPS
-
 Hypericum androsaemum

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
2.3.1.151 30
-
 assay at Hypericum androsaemum
2.3.1.220 35
-
-
 Hypericum androsaemum

Temperature Range [°C]

EC Number Temperature Minimum [°C] Temperature Maximum [°C] Comment Organism
2.3.1.151 35 40 optimal temperature Hypericum androsaemum

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
2.3.1.151 0.00685
-
 benzoyl-CoA mutant T135L Hypericum androsaemum
2.3.1.151 0.055
-
 benzoyl-CoA wild-type Hypericum androsaemum
2.3.1.220 0.0068
-
 malonyl-CoA pH 7.0, 35°C, mutant T135L enzyme Hypericum androsaemum
2.3.1.220 0.0068
-
 benzoyl-CoA pH 7.0, 35°C, mutant T135L enzyme Hypericum androsaemum
2.3.1.220 0.055
-
 malonyl-CoA pH 7.0, 35°C, wild-type enzyme Hypericum androsaemum
2.3.1.220 0.055
-
 benzoyl-CoA pH 7.0, 35°C, wild-type enzyme Hypericum androsaemum

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
2.3.1.151 7
-
 assay at Hypericum androsaemum
2.3.1.151 7.5
-
 optimal pH Hypericum androsaemum
2.3.1.220 6.5 7
-
 Hypericum androsaemum

pI Value

EC Number Organism Comment pI Value Maximum pI Value
2.3.1.220 Hypericum androsaemum
-
-
 5.78

General Information

EC Number General Information Comment Organism
2.3.1.220 evolution BPS is a type III polyketide synthase, PKS, and part of the chalcone synthase group of the superfamily of enzymes Hypericum androsaemum
2.3.1.220 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
2.3.1.220 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]

EC Number kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
2.3.1.151 6.414
-
 benzoyl-CoA wild-type Hypericum androsaemum
2.3.1.151 6.833
-
 benzoyl-CoA mutant T135L Hypericum androsaemum
2.3.1.220 0.0064
-
 malonyl-CoA pH 7.0, 35°C, wild-type enzyme Hypericum androsaemum
2.3.1.220 0.0064
-
 benzoyl-CoA pH 7.0, 35°C, wild-type enzyme Hypericum androsaemum
2.3.1.220 0.0068
-
 malonyl-CoA pH 7.0, 35°C, mutant T135L enzyme Hypericum androsaemum
2.3.1.220 0.0068
-
 benzoyl-CoA pH 7.0, 35°C, mutant T135L enzyme Hypericum androsaemum