3-Ketoacyl Synthases: Modification and Characterization

9728786个Jaworski 3-酮酰基合成酶(KAs)在多种代谢领域催化克莱森缩合反应。依赖丙二酰辅酶A和丙二酰基酰基载体蛋白(ACP)的KASS用于脂肪酸、聚酮和类黄酮的生物合成，在决定这些途径的最终产物方面起着重要作用。本课题要研究的两种酶分别是菠菜中的3-酮酰基-ACPIII(KAS I11)和拟南芥中的脂肪酸延伸酶KAS。KAS III利用乙酰辅酶A和丙二酰ACP催化植物和细菌合成脂肪酸的初始缩合反应。脂肪酸延伸酶KAS由FAE1编码，是一种利用丙二酰辅酶A的膜结合酶，参与油菜种子中20：1和22：1脂肪酸的合成。该项目的具体目标是：1.KAS III的定点突变。菠菜KAS III的高度保守的氨基酸残基将被中性氨基酸(如丙氨酸)或与保守残基相似的氨基酸取代。每取代一次，就会分析与这种缩合酶的S机理有关的部分反应。这些反应包括1.乙酰基-CoA结合，2.活性部位的乙酰化，2.丙二酰-ACP结合，4.丙二酰脱羧基，5.缩合，和6.乙酰转酰化(图3)。目的是确定高度保守的残基对KAS III机制每个部分的影响。2.脂肪酸拉伸酶KAS的工程与诱变通过用丙氨酸和丝氨酸替换保守的CyS来确认活性部位CyS的身份。改变与KAS III和查尔酮合酶保守的C末端区域残基的影响也将被确定。3.KAS三维结构的初步确定III.菠菜KAS Ill已在大肠杆菌中表达并纯化至均一。这种材料将被用来尝试生长晶体，如果成功，将确定KAS III的三维结构。KAS III和脂肪酸延伸酶KAS属于一类对初级和次级代谢有深刻影响的酶。在脂肪酸、聚酮和类黄酮的合成中，参与代谢的缩合酶是每条途径产物的主要决定因素之一。例如，海参、油菜和荷荷巴种子中的超长链脂肪酸的组成可以直接与每种植物中发现的脂肪酸延伸酶KAS的底物专一性有关。由于缩合酶的底物特异性在新陈代谢中起着如此关键的作用，因此阐明KASS的哪些结构特征影响这方面的酶活性是很重要的。然而，脂肪酸KASS催化的克莱森缩合的机制在所有情况下都知之甚少。只有少数相关的酶也能进行克莱森缩合，也就是说。硫解酶。

9728786 Jaworski 3-Ketoacyl synthases (KAS) catalyze Claissen condensation reactions in a variety of areas of metabolism. The malonyl-CoA and malonyl-acy carrier protein (ACP) dependent KASs utilized in fatty acid, polyketide and flavanoid biosynthesis play a major role in determining the final products of these pathways. The two enzymes to be studied in this project are 3-ketoacy1-ACPIII (KAS Ill) from spinach and fatty acid elongase KAS from Arabidopsis. KAS III catalyzes the initial condensation reaction of plant and bacterial fatty acid synthesis, utilizing acetyl-CoA and malonyl-ACP. The fatty acid elongase KAS encoded for by FAE1 is a membrane-bound enzyme that utilizes malonyl-CoA and is involved with 20:1 and 22:1 fatty acid synthesis in oil seeds. The specific objectives of the project are: 1. Site-directed mutagenesis of KAS III. Highly conserved amino acid residues of the spinach KAS Ill will be substituted either by neutral amino acids, e.g. alanine, or by amino acids similar to the conserved residue. With each substitution, the partial reactions relating to this condensing enzyme' s mechanism will be analyzed. These reactions include 1. acety1-CoA binding, 2. acetylation of the active site, 2. malonyl-ACP binding, 4. malonyl decarboxylation, 5. condensation, and 6. acetyl transacylation (Fig. 3). The objective is to determine the effect of highly conserved residues on each part of the KAS III mechanism. 2. Engineering and mutagenesis of fatty acid elongase KAS. The identity of the active site cys will be confirmed by substituting conserved cys with alanine and serine. The effect of altering residues in the C-terminus region that are conserved with KAS III and chalcone synthase will also be determined. 3. Initiate determination of the 3-D structure of KAS III. Spinach KAS Ill has been expressed in E. coli and purified to homogeneity. This material will be used in an attempt to grow crystals, and, if successful, determine the 3-dimensional structure of KAS III. Si gnificance. KAS III and the fatty acid elongase KAS belong to a class of enzymes that have profound effects on primary and secondary metabolism. In fatty acid, polyketide, and flavanoid synthesis, the condensing enzymes that take part in this metabolism are one the major determinants of the products of each pathway. For example, the composition of very long chain fatty acids in the seeds of A. thaliana, rape and jojoba can be directly related to the substrate specificity of the fatty acid elongase KAS found in each. Because the substrate specificities of condensing enzymes play such a key role in metabolism, it is important to elucidate which structural features of KASs affect this aspect of the enzymes' activities. However, the mechanisms for the Claissen condensation that fatty acid KASs catalyze are, in all cases, poorly understood. Limited information has been obtained for only a small number of related enzymes that also carry out Claissen condensations, viz. thiolases.