Kinetic analysis of the malonyl coenzyme A decarboxylation and the condensation reaction of fatty acid synthesis. Application to the study of malonyl coenzyme A inactivated chicken liver fatty acid synthetase

Srinivasan, K.R.; Kumar, Sunil

doi:10.1021/bi00515a015

Cited by 5 publications

(2 citation statements)

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“…A number of fatty acid synthases catalyze decarboxylation reactions without the active-site thiol being occupied by an enzyme-bound intermediate but at less than 25% of the product formation rate ( − ). In addition, substitutions of the active-site cysteine in the structurally homologous KAS II eliminate the condensation reaction but conserve decarboxylase activity ().…”

Section: Discussionmentioning

confidence: 99%

Dissection of Malonyl-Coenzyme A Decarboxylation from Polyketide Formation in the Reaction Mechanism of a Plant Polyketide Synthase

et al. 2000

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Chalcone synthase (CHS) catalyzes formation of the phenylpropanoid chalcone from one p-coumaroyl-CoA and three malonyl-coenzyme A (CoA) thioesters. The three-dimensional structure of CHS [Ferrer, J.-L., Jez, J. M., Bowman, M. E., Dixon, R. A., and Noel, J. P. (1999) Nat. Struct. Biol. 6, 775-784] suggests that four residues (Cys164, Phe215, His303, and Asn336) participate in the multiple decarboxylation and condensation reactions catalyzed by this enzyme. Here, we functionally characterize 16 point mutants of these residues for chalcone production, malonyl-CoA decarboxylation, and the ability to bind CoA and acetyl-CoA. Our results confirm Cys164's role as the active-site nucleophile in polyketide formation and elucidate the importance of His303 and Asn336 in the malonyl-CoA decarboxylation reaction. We suggest that Phe215 may help orient substrates at the active site during elongation of the polyketide intermediate. To better understand the structure-function relationships in some of these mutants, we also determined the crystal structures of the CHS C164A, H303Q, and N336A mutants refined to 1.69, 2.0, and 2.15 A resolution, respectively. The structure of the C164A mutant reveals that the proposed oxyanion hole formed by His303 and Asn336 remains undisturbed, allowing this mutant to catalyze malonyl-CoA decarboxylation without chalcone formation. The structures of the H303Q and N336A mutants support the importance of His303 and Asn336 in polarizing the thioester carbonyl of malonyl-CoA during the decarboxylation reaction. In addition, both of these residues may also participate in stabilizing the tetrahedral transition state during polyketide elongation. Conservation of the catalytic functions of the active-site residues may occur across a wide variety of condensing enzymes, including other polyketide and fatty acid synthases.

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Section: Discussionmentioning

confidence: 99%

Dissection of Malonyl-Coenzyme A Decarboxylation from Polyketide Formation in the Reaction Mechanism of a Plant Polyketide Synthase

et al. 2000

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“…These products were likely generated through spontaneous aldol cyclizations and intramolecular transesterification of the linear heptaketide intermediate 11 (Scheme 2A). Additionally, triacetic acid lactone ( 10 ) 15 was observed, a product of self-condensation from a truncated triketide intermediate (Scheme 2B). All of these side products were produced in significantly lower quantities when the PT or TE was present underscoring the central roles of these domains in both directed cyclization and overall catalytic efficiency.…”

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confidence: 99%