Engineered biosynthesis of plant polyketides: Chain length control in an octaketide-producing plant type III polyketide synthase
Engineered biosynthesis of plant polyketides: Chain length control in an octaketide-producing plant type III polyketide synthase
复制标题
DOI：
10.1021/ja053945v
复制
发表时间：
2005-09-14
影响因子：
15
通讯作者：
Noguchi, H
中科院分区：
文献类型：
--
作者：
Abe, I;Oguro, S;Noguchi, H
查尔酮合成酶(CHS)超家族的III型聚酮合成酶(PKS)产生多种植物次生代谢物，具有显著的结构多样性和生物活性(如查尔酮、二苯乙烯、二苯甲酮、丙烯酮、间苯三酚、间苯二酚、吡喃和色酮)。在这里，我们描述了一种由芦荟(Aloe Arboresens)产生的新型植物特异性III型PKS，其氨基酸序列与其他植物CHS超家族酶有50%-60%的同源性。在大肠杆菌中表达的重组酶催化丙二酰辅酶A连续七次脱羧缩合生成芳香族八肽SEK4和SEK4b，这是已知的由结构简单的III型PKS合成的最长的聚酮。令人惊讶的是，定点突变揭示了单个残基Gly207(对应于CHS的活性位点Thr197)决定了聚酮链的长度和产物的特异性。从小到大的取代(G207A、G207T、G207M、G207L、G207F和G207W)导致八肽形成活性的丧失，并伴随着较短链长的多酮(从三酮到七肽)的形成，包括五酮色酮、2，7-二羟基-5-甲基色酮和六酮吡喃酮，6-(2，4-dihydroxy-6-methylphenyl)-4-hydroxy-2-pyrone，取决于侧链的大小。值得注意的是，III型PKS的功能多样性被证明是从活性中心腔内化学惰性单一残基的简单空间调节以及Cys-His-Asn催化三联体的保守进化而来的。这为具有重要药用价值的植物多酮的工程生物合成提供了新的策略。
The chalcone synthase (CHS) superfamily of type III polyketide synthases (PKSs) produces a variety of plant secondary metabolites with remarkable structural diversity and biological activities (e.g., chalcones, stilbenes, benzophenones, acrydones, phloroglucinols, resorcinols, pyrones, and chromones). Here we describe an octaketide-producing novel plant-specific type III PKS from aloe (Aloe arborescens) sharing 50-60% amino acid sequence identity with other plant CHS-superfamily enzymes. A recombinant enzyme expressed in Escherichia coli catalyzed seven successive decarboxylative condensations of malonyl-CoA to yield aromatic octaketides SEK4 and SEK4b, the longest polyketides known to be synthesized by the structurally simple type III PKS. Surprisingly, site-directed mutagenesis revealed that a single residue Gly207 (corresponding to the CHS's active site Thr197) determines the polyketide chain length and product specificity. Small-to-large substitutions (G207A, G207T, G207M, G207L, G207F, and G207W) resulted in loss of the octaketide-forming activity and concomitant formation of shorter chain length polyketides (from triketide to heptaketide) including a pentaketide chromone, 2,7-dihydroxy-5-methylchromone, and a hexaketide pyrone, 6-(2,4-dihydroxy-6-methylphenyl)-4-hydroxy-2-pyrone, depending on the size of the side chain. Notably, the functional diversity of the type III PKS was shown to evolve from simple steric modulation of the chemically inert single residue lining the active-site cavity accompanied by conservation of the Cys-His-Asn catalytic triad. This provided novel strategies for the engineered biosynthesis of pharmaceutically important plant polyketides.