Study on the Alteration of Catalytic Activities by the Change of Three Dimensional Structure of Enzyme Proteins

查尔酮合成酶(CHS)是植物来源的具有代表性的聚酮合成酶，催化类黄酮生物合成的第一反应形成其基本骨架。CHS广泛存在于植物体内，对保护植物免受外界胁迫具有重要的生理作用。与查尔酮合酶相关的其他聚酮合酶具有很高的同源性，称为查尔酮合酶超家族。CHS反应与脂肪酸合成酶(Fas)非常相似，但其独特的酶反应是丙二酰辅酶A与脱羧基的直接缩合，而不是脂肪酸合成酶中的ACP丙二酰辅酶A。CHS的小分子量(约43KD)是最吸引人的研究热点。1.建立了大肠杆菌、酵母和空泡病毒的异源表达系统，获得了大量的CHESS，并对不同表达系统获得的CHIS的反应进行了详细的研究。2.首次证明了查尔酮合酶和二苯乙烯合酶存在交叉反应，即这两种酶产生的副产物是对偶酶产物。当这些酶在真核细胞系统中表达时，交叉反应程度降低。3.裸子植物是最原始的维管束植物，能产生苯基吡喃酮。获得了CHIS超家族的5个克隆，并对它们的反应进行了表征。除了CHIS、两个STS和异戊酮合成酶外，它还含有不起作用的CHIS家族mRNA。聚酮合成酶中的必需组氨酸被谷氨酰胺和突变体取代，突变的谷氨酰胺为组氨酸，与CHIS和STS一样，聚酮合成酶没有发生任何明显的反应。酶活性的缺乏是由于整个三维结构的变化，而这种变化又受到其他氨基酸变化的影响。

Chalcone synthase (CHS) is the representative polyketide synthase of plant origin, which catalyzes the first reaction of flavonoid biosynthesis to form its basic skeleton. CHSs are ubiquitously present in plants and play essential physiological role to protect plants from outer stresses. The other polyketide synthases relating to chalcone synthase have a high homology and called chalcone synthase superfamily. The reaction CHS is very similar to fatty acid synthase (FAS), however its unique enzyme reaction is the direct condensation of malonyl CoA with decarboxylation in contrast to ACP malonyl CoA in FAS. Small molecular weight of CHS (ca. 43KD) is the most attractive point of study. 1. Heterologous expression sytems with E. coli, yeast and vaculovirus were established to obtain Chess in a large amount and reactions of CHIS obtained by different expression systems were investigated in details. 2. We first demonstrate that chalcone and stilbene synthases showed cross reactions, that is the two enzyme produced counter part enzyme products as by products. The degree of cross reaction was decreased when the enzymes were expressed in eukaryote systems. 3. Psilotum nodum is the most primitive vascular plant which produces phnylpyrone. Five clones of CHIS superfamily were obtained and their reaction was characterized. In addition to CHIS, two STS and isovalerophenone synthase it contains unfunctional CHIS family mRNA. Essential histidine in polyketide synthase is replaced with glutamine and revertant, mutated glutamine to histidine as in CHIS and STS did not show any significant reaction of polyketide synthase. The lack of enzyme activity is due to the change of whole three dimentional structure which is affected by the alteration of other amino acids.