Jez J M, Ferrer J L, Bowman M E, Dixon R A, Noel J P
Structural Biology Laboratory, The Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla, California 92037, USA.
Biochemistry. 2000 Feb 8;39(5):890-902. doi: 10.1021/bi991489f.
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.
查尔酮合酶（CHS）催化由一分子对香豆酰辅酶A和三分子丙二酰辅酶A硫酯形成苯丙素类查尔酮。CHS的三维结构[费雷尔，J.-L.，杰兹，J. M.，鲍曼，M. E.，迪克森，R. A.，以及诺埃尔，J. P.（1999年）《自然结构生物学》6，775 - 784]表明四个残基（半胱氨酸164、苯丙氨酸215、组氨酸303和天冬酰胺336）参与了该酶催化的多重脱羧和缩合反应。在此，我们对这些残基的16个点突变体进行了功能表征，涉及查尔酮生成、丙二酰辅酶A脱羧以及结合辅酶A和乙酰辅酶A的能力。我们的结果证实了半胱氨酸164在聚酮化合物形成中作为活性位点亲核试剂的作用，并阐明了组氨酸303和天冬酰胺336在丙二酰辅酶A脱羧反应中的重要性。我们认为苯丙氨酸215可能在聚酮化合物中间体延长过程中有助于在活性位点定向底物。为了更好地理解其中一些突变体的结构 - 功能关系，我们还分别测定了CHS C164A、H303Q和N336A突变体的晶体结构，分辨率分别细化到1.69埃、2.0埃和2.15埃。C164A突变体的结构表明，由组氨酸303和天冬酰胺336形成的假定氧阴离子洞保持不变，使得该突变体能够催化丙二酰辅酶A脱羧而不形成查尔酮。H303Q和N336A突变体的结构支持了组氨酸303和天冬酰胺336在脱羧反应中使丙二酰辅酶A硫酯羰基极化的重要性。此外，这两个残基在聚酮化合物延长过程中可能还参与稳定四面体过渡态。活性位点残基催化功能的保守性可能存在于多种缩合酶中，包括其他聚酮化合物和脂肪酸合酶。
