Cloned (Comment) | Organism |
---|---|
expression of recombinant His-tagged thioesterase/Claisen cyclase in Escherichia coli, expression of the selenomethionine-labeled PksA TE in Escherichia coli methionine auxotroph strain B834(DE3) | Aspergillus sp. |
Crystallization (Comment) | Organism |
---|---|
purified recombinant selenomethionine-labeled of PksA TE, sitting drop vapour diffusion method, 5 mg/ml protein in 20 mM Tris-HCl pH 7.5 containing 5% glycerol and 2 mM DTT is mixed with well solution containing 0.2 M ammonium acetate, 0.1 M sodium citrate pH 5.6, and 30% PEG 4000, 25°C, 2 days, X-ray diffraction structure determination and analysis, modeling | Aspergillus sp. |
Protein Variants | Comment | Organism |
---|---|---|
D1964N | site-directed mutagenesis of a catalytic residue | Aspergillus sp. |
D2070N | site-directed mutagenesis, the mutation results in only a slightly reduced rate of hydrolysis compared to the apo-mutant | Aspergillus sp. |
H2088F | site-directed mutagenesis of a catalytic residue | Aspergillus sp. |
S1937A | site-directed mutagenesis of a catalytic residue | Aspergillus sp. |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
7 malonyl-CoA + hexanoyl-[acyl-carrier protein] | Aspergillus sp. | - |
7 CoA + norsolorinic acid anthrone + [acyl-carrier protein] + 7 CO2 + 2 H2O | - |
? |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Aspergillus sp. | Q12052 | - |
- |
Purification (Comment) | Organism |
---|---|
recombinant His-tagged thioesterase/Claisen cyclase and selenomethionine-labeled PksA TE from Escherichia coli | Aspergillus sp. |
Reaction | Comment | Organism | Reaction ID |
---|---|---|---|
7 malonyl-CoA + hexanoyl-[acyl-carrier protein] = 7 CoA + norsolorinic acid anthrone + [acyl-carrier protein] + 7 CO2 + 2 H2O | mechanism of thioesterase/Claisen cyclase-catalyzed chain-termination of fungal aromatic polyketide biosynthesis. The ACP of the ACP-bound substrate is displaced upon thioesterase-catalyzed transesterification. Rotation of the substrate side chain can occur once the ACP leaves the pocket, and the thioesterase can then close. Thioesterase conformational constraints as observed in the closed-form crystal structure guide Claisen-type cyclization to release noranthrone, i.e. norsolorinic acid anthrone, the polyketide precursor of aflatoxin B1. Domain structure and reaction mechanism, detailed overview | Aspergillus sp. |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
7 malonyl-CoA + hexanoyl-[acyl-carrier protein] | - |
Aspergillus sp. | 7 CoA + norsolorinic acid anthrone + [acyl-carrier protein] + 7 CO2 + 2 H2O | - |
? |
Subunits | Comment | Organism |
---|---|---|
More | domain architecture, the enzyme shows an alpha/beta-hydrolase fold in the catalytic closed form with a distinct hydrophobic substrate-binding chamber involving the PksA thioesterase/Claisen cyclase residues Ser1937, His2088, and Asp1964, which constitute the catalytic triad conserved in the alpha/beta-hydrolase family, detailed overview | Aspergillus sp. |
Synonyms | Comment | Organism |
---|---|---|
PksA | - |
Aspergillus sp. |
polyketide synthase A | - |
Aspergillus sp. |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
25 | - |
assay at | Aspergillus sp. |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
7.5 | - |
assay at | Aspergillus sp. |
General Information | Comment | Organism |
---|---|---|
metabolism | polyketide synthase A is a multidomain PKS central to the biosynthesis of aflatoxin B1, a potent environmental carcinogen | Aspergillus sp. |
additional information | the synthetic versatility of thioesterase domains in fungal nonreducing, iterative PKSs extends to Claisen cyclase chemistry by catalyzing C-C ring closure reactions as opposed to thioester hydrolysis or O-C/N-C macrocyclization observed in other thioesterase structures. Catalysis of C-C bond formation as a product release mechanism dramatically expands the synthetic potential of PKSs, structural analyses of the thioesterase/CLC domain in polyketide synthase A | Aspergillus sp. |