3-Ketoacyl Synthases: Modification and Characterization

9728786 Jaworski 3-酮酰基脱氢酶（KAS）在多种代谢领域催化Claissen缩合反应。在脂肪酸、聚酮化合物和类黄烷酸的生物合成中，丙二酰辅酶A和丙二酰酰基载体蛋白（ACP）依赖的KAS在决定这些途径的最终产物中起主要作用。在这个项目中要研究的两种酶是来自菠菜的3-ketoacy 1-ACPIII（KAS III）和来自拟南芥的脂肪酸延伸酶KAS。KAS III利用乙酰辅酶A和丙二酰-ACP催化植物和细菌脂肪酸合成的初始缩合反应。由FAE 1编码的脂肪酸延伸酶KAS是一种膜结合酶，其利用丙二酰辅酶A并参与油籽中的20：1和22：1脂肪酸合成。该项目的具体目标是：1。KAS III的定点突变。菠菜KAS III的高度保守氨基酸残基将被中性氨基酸例如丙氨酸或与保守残基相似的氨基酸取代。对于每一个取代，将分析与该缩合酶的机制有关的部分反应。这些反应包括1。乙酰基-CoA结合，2.活性位点的乙酰化，2.丙二酰-ACP结合，4.丙二酰脱羧，5.冷凝，和6.乙酰基转酰作用（图3）。目的是确定高度保守残基对KAS III机制各部分的影响。2.脂肪酸延伸酶KAS的工程改造和诱变。活性位点cys的身份将通过用丙氨酸和丝氨酸取代保守的cys来确认。还将确定改变与KAS III和查耳酮合酶保守的C-末端区域中的残基的影响。3.开始确定KAS III的三维结构。菠菜KAS III已在E. coli中，并纯化至均一。这种材料将用于尝试生长晶体，如果成功，确定KAS III的三维结构。意义重大。KAS III和脂肪酸延伸酶KAS属于一类对初级和次级代谢具有深远影响的酶。在脂肪酸、聚酮化合物和类黄烷化合物的合成中，参与这种代谢的缩合酶是每种途径产物的主要决定因素之一。例如，A.拟南芥、油菜和霍霍巴的脂肪酸延伸酶的底物特异性可与在每种中发现的脂肪酸延伸酶KAS的底物特异性直接相关。由于缩合酶的底物特异性在代谢中起着关键作用，因此阐明KAS的结构特征影响酶活性的这一方面是很重要的。然而，在所有情况下，对脂肪酸KAS催化的Claissen缩合的机制知之甚少。有限的信息已经获得了只有少数相关的酶，也进行克莱森缩合，即硫解酶。

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.