tailieunhanh - Báo cáo Y học: Studies into factors contributing to substrate specificity of membrane-bound 3-ketoacyl-CoA synthases

We are interested in constructing a model for the substrate-bindingsiteof fattyacidelongase-13-ketoacylCoAsynthase (FAE1 KCS),the enzyme responsible forproductionof very long chain fatty acids of plant seed thaliana andBrassica napusFAE1 KCS enzymes are highly homo-logous but the seed oil content of these plants suggests that their substrate specificities differ with respect to acyl chain length. | Eur. J. Biochem. 269 4789-4798 22002 FEBS 2002 doi Studies into factors contributing to substrate specificity of membrane-bound 3-ketoacyl-CoA synthases Brenda J. Blacklock and Jan G. Jaworski Department of Chemistry and Biochemistry Miami University Oxford Ohio USA We are interested in constructing a model for the substratebinding site of fatty acid elongase-1 3-ketoacyl CoA synthase FAE1 KCS dhe enzyme le 1 peon ll bl k for production of very long chain fatty acids of plant seed oils. Arabidopsis thaliana and Brassica napus FAE1 KCS enzymes are highly homologous but the seed oil content of these plants suggests that their substrate specificities differ with respect to acyl chain length. We used in vivo and in vitro assays of Saccharomyces cerevisiae-expressed FAE1 KCSs to demonstrate that the B. napus FAE1 KCS enzyme favors longer chain acyl substrates than the A. thaliana enzyme. Domains residues responsible for substrate specificity were investigated by determining catalytic activity and substrate specificity of chimeric enzymes of A. thaliana and B. napus FAE1 KCS. The N-terminal region exudining the a ammembrane domain was shown to be involved in substrate specificity. One chimeric enzyme that included A. thaliam sequence from the N terminus to residue 114 and B. napus sequence from residue 115 to the C terminus had substrate specificity similar to that of A. thaliana FAE1 KCS. However a K92R substitution in this chimeric enzyme changed the specificity to that of the B. napus enzyme without loss of catalytic activity. Thus this study was successful in identifying a domain involved in determining substrate specificity in FAE1 KCS and in engineering an enzyme with novel activity. Keywords Arabidopsis thaliana Brassica napus fatty acid elongation 3-ketoacyl-CoA synthase. The very long chain fatty acids VLCFA found in seed oils are derived from the elongation of products of de novo fatty acid biosynthesis 1 . The initial reaction