Jm. Jez et al., Dissection of malonyl-coenzyme A decarboxylation from polyketide formationin the reaction mechanism of a plant polyketide synthase, BIOCHEM, 39(5), 2000, pp. 890-902
Chalcone synthase (CHS) catalyzes formation of the phenylpropanoid chalcone
from one p-coumaroyl-CoA and three malonyl-coenzyme A (CoA) thioesters. Th
e three-dimensional structure of CHS [Ferrer, J.-L., Jet, J. M., Bowman, M.
E., Dixon, R. A., and Noel, J. P. (1999) Nat. Struct. Biol. 6, 775-784] su
ggests that four residues (Cys164, Phe215, His303, and Asn336) participate
in the multiple decarboxylation and condensation reactions catalyzed by thi
s enzyme. Here, we functionally characterize 16 point mutants of these resi
dues 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 H
is303 and Asn336 in the malonyl-CoA decarboxylation reaction. We suggest th
at Phe215 may help orient substrates at the active site during elongation o
f the polyketide intermediate. To better understand the structure-function
relationships in some of these mutants, we also determined the crystal stru
ctures of the CHS C164A, H303Q, and N336A mutants refined to 1.69, 2.0, and
2.15 Angstrom 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 s
upport the importance of His303 and Asn336 in polarizing the thioester carb
onyl of malonyl-CoA during the decarboxylation reaction. In addition, both
of these residues may also participate in stabilizing the tetrahedral trans
ition state during polyketide elongation. Conservation of the catalytic fun
ctions of the active-site residues may occur across a wide variety of conde
nsing enzymes, including other polyketide and fatty acid synthases.