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Literature summary for 2.3.1.221 extracted from

  • Korman, T.P.; Crawford, J.M.; Labonte, J.W.; Newman, A.G.; Wong, J.; Townsend, C.A.; Tsai, S.C.
    Structure and function of an iterative polyketide synthase thioesterase domain catalyzing Claisen cyclization in aflatoxin biosynthesis (2010), Proc. Natl. Acad. Sci. USA, 107, 6246-6251.
    View publication on PubMedView publication on EuropePMC

Cloned(Commentary)

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 (Commentary)

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

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/ Products (Substrates)

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

Organism UniProt Comment Textmining
Aspergillus sp. Q12052
-
-

Purification (Commentary)

Purification (Comment) Organism
recombinant His-tagged thioesterase/Claisen cyclase and selenomethionine-labeled PksA TE from Escherichia coli Aspergillus sp.

Reaction

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 and Products (Substrate)

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

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

Synonyms Comment Organism
PksA
-
Aspergillus sp.
polyketide synthase A
-
Aspergillus sp.

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
25
-
assay at Aspergillus sp.

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
7.5
-
assay at Aspergillus sp.

General Information

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.