Structural control of polyketide formation in plant-specific polyketide synthases
Structural control of polyketide formation in plant-specific polyketide synthases
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DOI：
10.1016/s1074-5521(00)00041-7
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发表时间：
2000-12-01
影响因子：
--
通讯作者：
Noel, JP
中科院分区：
文献类型：
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作者：
Jez, JM;Austin, MB;Noel, JP
背景：聚酮合成酶(PKS)通过利用不同的起始分子和控制聚酮的最终长度来产生分子多样性。尽管利用这种机制的可变性已经产生了新的聚酮，但这种多功能性的结构基础尚不清楚。植物特有的PKS对于抗微生物植物保卫素、花青素花色素和根瘤菌结瘤基因的诱导子的生物合成是必不可少的。2-吡喃酮合酶(2-PS)和查尔酮合酶(CHS)是植物特有的PKS，氨基酸序列同源性为74%。2-PS由一个乙酰辅酶A起始分子和两个丙二酰辅酶A形成三酮基甲基吡喃酮。CHS使用一个p-香豆酰辅酶A起始分子和三个丙二酰辅酶A来生产四酮查尔酮。我们的目的是阐明这类PKS起始分子选择性和聚酮长度控制的分子基础。结果：通过分子置换确定了2-PS与反应中间体乙酰乙酰辅酶A络合的2.05埃分辨晶体结构。2-PS和CHS具有共同的三维折叠、一组保守的催化残基和相似的CoA结合位点。但是，2-PS的活性位空穴小于CHS的活性位空穴。在2-PS起始/延伸空腔内的28个残基中，CHS中有4个位置不同，CHS中其中3个位置(T197L、G256L和S338I)的点突变改变了产物的形成。将这些突变组合到CHS三重突变体(T197L/G256L/S3381)中，产生了一种与2-PS功能相同的酶。结论：2-PS的结构和功能特征以及具有类似2-PS的起始/延伸空腔的CHS突变体的产生表明空腔体积影响起始分子的选择，并控制聚酮的最终长度。这些结果为控制其他PKS中的聚酮长度提供了结构基础，并为进一步扩大聚酮生物合成多样性的范围提供了策略。
Background: Polyketide synthases (PKSs) generate molecular diversity by utilizing different starter molecules and by controlling the final length of the polyketide. Although exploitation of this mechanistic variability has produced novel polyketides, the structural foundation of this versatility is unclear. Plant-specific PKSs are essential for the biosynthesis of anti-microbial phytoalexins, anthocyanin floral pigments, and inducers of Rhizobium nodulation genes. 2-Pyrone synthase (2-PS) and chalcone synthase (CHS) are plant-specific PKSs that share 74% amino acid sequence identity. 2-PS forms the triketide methylpyrone from an acetyl-CoA starter molecule and two malonyl-CoAs. CHS uses a p-coumaroyl-CoA starter molecule and three malonyl-CoAs to produce the tetraketide chalcone. Our goal was to elucidate the molecular basis of starter molecule selectivity and control of polyketide length in this class of PKS.Results: The 2.05 Angstrom resolution crystal structure of 2-PS complexed with the reaction intermediate acetoacetyl-CoA was determined by molecular replacement. 2-PS and CHS share a common three-dimensional fold, a set of conserved catalytic residues, and similar CoA binding sites. However, the active site cavity of 2-PS is smaller than the cavity in CHS. Of the 28 residues lining the 2-PS initiation/elongation cavity, four positions vary in CHS, Point mutations at three of these positions in CHS (T197L, G256L, and S338I) altered product formation. Combining these mutations in a CHS triple mutant (T197L/G256L/S3381) yielded an enzyme that was functionally identical to 2-PS.Conclusions: Structural and functional characterization of 2-PS together with generation of a CHS mutant with an initiation/elongation cavity analogous to 2-PS demonstrates that cavity volume influences the choice of starter molecule and controls the final length of the polyketide. These results provide a structural basis for control of polyketide length in other PKSs, and suggest strategies for further increasing the scope of polyketide biosynthetic diversity.