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
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 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 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
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
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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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