A multi-domain polyketide synthase involved in the synthesis of aflatoxins in the fungus Aspergillus parasiticus. The hexanoyl starter unit is provided to the acyl-carrier protein (ACP) domain by a dedicated fungal fatty acid synthase .
The enzyme appears in viruses and cellular organisms
reaction mechanism, overview. The SAT domain in PksA selects a hexanoyl starter unit. The MAT domain loads the free ACP with malonyl units. After seven successive condensation events with malonyl-ACP catalysed in te ketoacyl synthase domain, the linear ACP-bound polyketide is cyclized (C4-C9 and C2-C11 cyclization events) and aromatized in the product template domain to give the bicyclic intermediate. The thioesterase domain catalyses C-C cyclization to release anthrone, which undergoes oxidation to the anthraquinone norsolorinic acid, to initiate the complex biosynthetic pathway to aflatoxin B1
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
a product template domain unites with the ketosynthase and thioesterase in this IPKS system to assemble precisely seven malonyl-derived building blocks to a hexanoyl starter unit and mediate a specific cyclization cascade. These mechanistic features are general for IPKS-catalyzed production of aromatic polyketides
A multi-domain polyketide synthase involved in the synthesis of aflatoxins in the fungus Aspergillus parasiticus. The hexanoyl starter unit is provided to the acyl-carrier protein (ACP) domain by a dedicated fungal fatty acid synthase [1].
Substrates: processivity of polyketide extension and substrate specificity, overview. The enzyme shows activity against hexanoyl- and acetyl-, but not malonyl-CoA. Rapid loading of extension units onto the carrier domain facilitates efficient chain extension in a manner kinetically favorable to ultimate product formation. Essential roles of the product template and thioesterase domains for cyclization and product release, editing role for the thioesterase domain Products: -
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7 malonyl-CoA + hexanoyl-[acyl-carrier protein]
7 CoA + norsolorinic acid anthrone + [acyl-carrier protein] + 7 CO2 + 2 H2O
the pksA gene is located in the aflatoxin pathway gene cluster and is linked to the nor-1 gene, an aflatoxin pathway gene required for converting norsolorinic acid to averantin
the pksA gene is located in the aflatoxin pathway gene cluster and is linked to the nor-1 gene, an aflatoxin pathway gene required for converting norsolorinic acid to averantin
the enzyme also has an editing function for the C-terminal thioesterase domain beyond its synthetic role in Claisen/Dieckmann cyclization and product release. Domain architecture and expected enzyme-bound intermediates of PksA, overview
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
PksA domain structure, from N- to C-terminus including the starter unit: acylcarrier protein transacylase (SAT), beta-ketoacyl synthase (KS), malonyl-CoA: acyl-carrier protein transacylase (MAT), product template (PT), acyl-carrier protein (ACP), and thioesterase/Claisen cyclase (TE/CLC)
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
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CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
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
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)
gene pksA, cloning of various mono-, di-, and tridomains for combinatorial reconstitution experiments, expression of the either C- or N-terminally His6-tagged protein fragments in Escherichia coli
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