1d6h

CHALONE SYNTHASE (N336A MUTANT COMPLEXED WITH COA)

Overview

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.

About this Structure

1D6H is a Single protein structure of sequence from Medicago sativa with SO4 and COA as ligands. Active as Naringenin-chalcone synthase, with EC number 2.3.1.74 Full crystallographic information is available from OCA.

Reference

Dissection of malonyl-coenzyme A decarboxylation from polyketide formation in the reaction mechanism of a plant polyketide synthase., Jez JM, Ferrer JL, Bowman ME, Dixon RA, Noel JP, Biochemistry. 2000 Feb 8;39(5):890-902. PMID:10653632

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