Dissection of malonyl-coenzyme A decarboxylation from polyketide formation in the reaction mechanism of a plant polyketide synthase

被引:279
作者
Jez, JM
Ferrer, JL
Bowman, ME
Dixon, RA
Noel, JP
机构
[1] Salk Inst Biol Studies, Struct Biol Lab, La Jolla, CA 92037 USA
[2] Samuel Roberts Noble Fdn Inc, Div Plant Biol, Ardmore, OK 73402 USA
关键词
D O I
10.1021/bi991489f
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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., Jet, 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 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 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.
引用
收藏
页码:890 / 902
页数:13
相关论文
共 85 条
  • [81] TENEYCK LF, 1973, ACTA CRYSTALLOGR A, VA 29, P183, DOI 10.1107/S0567739473000458
  • [82] EFFICIENT STRUCTURE-FACTOR CALCULATION FOR LARGE MOLECULES BY FAST FOURIER-TRANSFORM
    TENEYCK, LF
    [J]. ACTA CRYSTALLOGRAPHICA SECTION A, 1977, 33 (MAY1): : 486 - 492
  • [83] REACTION-MECHANISMS OF HOMODIMERIC PLANT POLYKETIDE SYNTHASES (STILBENE AND CHALCONE SYNTHASE) - A SINGLE ACTIVE-SITE FOR THE CONDENSING REACTION IS SUFFICIENT FOR SYNTHESIS OF STILBENES, CHALCONES, AND 6'-DEOXYCHALCONES
    TROPF, S
    KARCHER, B
    SCHRODER, G
    SCHRODER, J
    [J]. JOURNAL OF BIOLOGICAL CHEMISTRY, 1995, 270 (14) : 7922 - 7928
  • [84] Conversion of a β-ketoacyl synthase to a malonyl decarboxylase by replacement of the active-site cysteine with glutamine
    Witkowski, A
    Joshi, AK
    Lindqvist, Y
    Smith, S
    [J]. BIOCHEMISTRY, 1999, 38 (36) : 11643 - 11650
  • [85] Synthesis and structure-activity relationships of carboxylated chalcones: A novel series of CysLT(1) (LTD(4)) receptor antagonists
    Zwaagstra, ME
    Timmerman, H
    Tamura, M
    Tohma, T
    Wada, Y
    Onogi, K
    Zhang, MQ
    [J]. JOURNAL OF MEDICINAL CHEMISTRY, 1997, 40 (07) : 1075 - 1089