Structure-guided programming of polyketide chain-length determination in chalcone synthase
Structure-guided programming of polyketide chain-length determination in chalcone synthase
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DOI：
10.1021/bi015621z
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发表时间：
2001-12-11
期刊：
影响因子：
2.9
通讯作者：
Noel, JP
中科院分区：
文献类型：
--
作者：
Jez, JM;Bowman, ME;Noel, JP
查耳酮合酶 (CHS) 属于 III 型聚酮化合物合酶 (PKS) 家族，可催化结构多样的聚酮化合物的形成。 CHS 通过将丙二酰辅酶 A 脱羧衍生的三个乙酰基阴离子顺序缩合成连接到活性位点半胱氨酸的对香豆酰基部分来合成四酮化合物。 Gly256 位于 CHS 活性位点表面，与丙二酰辅酶 A 衍生的聚酮化合物链直接接触。因此，位置256作为理想位置。旨在探讨 III 型 PKS 中空腔体积与聚酮化合物链长测定之间的联系。 CHS G256A、G256V、G256L 和 G256F 突变体的功能检查揭示了与野生型 CHS 不同的产物谱。以对香豆酰辅酶 A 作为起始分子，G256A 和 G256V 突变体产生显着更多的四酮内酯。对空腔体积的进一步限制（例如在 G256L 和 G256F 突变体中所见）会导致来自三酮化合物中间体的苯乙烯吡喃酮双去甲仰光水平增加。 CHS G256A、G256V、G256L 和 G256F 突变体的 X 射线晶体结构证实，这些取代减少了活性位点空腔的大小，而多肽主链的构象没有显着改变。 256位侧链体积影响聚酮化合物扩链过程中缩合反应的数量以及环化反应过程中三酮化合物和四酮化合物中间体的构象。结合残基 256 的天然序列变异观察到的这些结果表明，通过位置 256 侧链体积的位点特异性进化，可以发生产物特异性的快速多样化，而不会同时损失相关 CHS 样酶的大量催化活性。
Chalcone synthase (CHS) belongs to the family of type III polyketide synthases (PKS) that catalyze formation of structurally diverse polyketides. CHS synthesizes a tetraketide by sequential condensation of three acetyl anions derived from malonyl-CoA decarboxylation to a p-coumaroyl moiety attached to an active site cysteine. Gly256 resides on the surface of the CHS active site that is in direct contact with the polyketide chain derived from malonyl-CoA. Thus, position 256 serves as an ideal. target to probe the link between cavity volume and polyketide chain-length determination in type III PKS. Functional examination of CHS G256A, G256V, G256L, and G256F mutants reveals altered product profiles from that of wild-type CHS. With p-coumaroyl-CoA as a starter molecule, the G256A and G256V mutants produce notably more tetraketide lactone. Further restrictions in cavity volume such as that seen in the G256L and G256F mutants yield increasing levels of the styrylpyrone bis-noryangonin from a triketide intermediate. X-ray crystallographic structures of the CHS G256A, G256V, G256L, and G256F mutants establish that these substitutions reduce the size of the active site cavity without significant alterations in the conformations of the polypeptide backbones. The side chain volume of position 256 influences both the number of condensation reactions during polyketide chain extension and the conformation of the triketide and tetraketide intermediates during the cyclization reaction. These results viewed in conjunction with the natural sequence variation of residue 256 suggest that rapid diversification of product specificity without concomitant loss of substantial catalytic activity in related CHS-like enzymes can occur by site-specific evolution of side chain volume at position 256.