3-Ketoacyl Synthases: Modification and Characterization

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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.