REDESIGN OF SOLUBLE FATTY-ACID DESATURASES FROM PLANTS FOR ALTERED SUBSTRATE-SPECIFICITY AND DOUBLE-BOND POSITION

Acyl-acyl carrier protein (ACP) desaturases introduce double bonds at specific positions in fatty acids of defined chain lengths and are one of the major determinants of the monounsaturated fatty acid compositi on of vegetable oils, Mutagenesis studies were conducted to determine the structural basis for the substrate and double bond positional spec ificities displayed by acyl-ACP desaturases. By replacement of specifi c amino acid residues in a Delta(6)-palmitoyl (16:0)-ACP desaturase wi th their equivalents from a Delta(9)-stearoyl (18:0)-ACP desaturase, m utant enzymes were identified that have altered fatty acid chain-lengt h specificities or that can insert double bonds into either the Delta( 6) or Delta 9 positions of 16:0- and 18:0-ACP. Most notably, by replac ement of five amino acids (A181T/A200F/S205N/L206T/G207A), the Delta(6 )-16:0-ACP desaturase was converted into an enzyme that functions prin cipally as a Delta(9)-18:0-ACP desaturase. Many of the determinants of fatty acid chain-length specificity in these mutants are found in res idues that line the substrate binding channel as revealed by x-ray cry stallography of the Delta(9)-18:0-ACP desaturase. The crystallographic model of the active site is also consistent with the diverged activit ies associated with naturally occurring variant acyl-ACP desaturases, In addition, on the basis of the active-site model, a Delta(9)-18:0-AC P desaturase was converted into an enzyme with substrate preference fo r 16:0-ACP by replacement of two residues (L118F/P179I), These results demonstrate the ability to rationally modify acyl-ACP desaturase acti vities through site-directed mutagenesis and represent a first step to ward the design of acyl-ACP desaturases for the production of novel mo nounsaturated fatty acids in transgenic oilseed crops.