Jez J M, Bowman M E, Noel J P
Structural Biology Laboratory, The Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla, California 92037, USA.
Biochemistry. 2001 Dec 11;40(49):14829-38. doi: 10.1021/bi015621z.
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.
查尔酮合酶（CHS）属于III型聚酮合酶（PKS）家族，该家族催化结构多样的聚酮化合物的形成。CHS通过将源自丙二酰辅酶A脱羧的三个乙酰阴离子顺序缩合到连接至活性位点半胱氨酸的对香豆酰部分上，合成一种四酮化合物。Gly256位于CHS活性位点的表面，该位点与源自丙二酰辅酶A的聚酮化合物链直接接触。因此，256位是探究III型PKS中空腔体积与聚酮化合物链长度确定之间联系的理想靶点。对CHS G256A、G256V、G256L和G256F突变体的功能检测显示，其产物谱与野生型CHS不同。以对香豆酰辅酶A作为起始分子时，G256A和G256V突变体产生的四酮内酯明显更多。如在G256L和G256F突变体中所见，对空腔体积的进一步限制会使三酮中间体产生的苯乙烯基吡喃双去甲氧基姜黄素水平增加。CHS G256A、G256V、G256L和G256F突变体的X射线晶体学结构表明，这些取代减小了活性位点空腔的大小，而多肽主链的构象没有明显改变。256位的侧链体积既影响聚酮化合物链延伸过程中的缩合反应次数，也影响环化反应过程中三酮和四酮中间体的构象。结合256位残基的天然序列变异来看，这些结果表明，在相关的类CHS酶中，通过256位侧链体积的位点特异性进化，可以在不伴随大量催化活性丧失的情况下实现产物特异性的快速多样化。
