A DETERMINANT OF SUBSTRATE-SPECIFICITY PREDICTED FROM THE ACYL-ACYL CARRIER PROTEIN DESATURASE OF DEVELOPING CATS CLAW SEED

Cat's claw (Doxantha unguis-cati L.) vine accumulates nearly 80% palmi toleic acid (16:1 Delta 9) plus cis-vaccenic acid (18:1 Delta 11) in i ts seed oil. To characterize the biosynthetic origin of these unusual fatty acids, cDNAs for acyl-acyl carrier protein (acyl-ACP) desaturase s were isolated from developing cat's claw seeds. The predominant acyl -ACP desaturase cDNA identified encoded a polypeptide that is closely related to the stearoyl (Delta 9-18:0)-ACP desaturase from castor (Ric inis communis L.) and other species. Upon expression in Escherichia co li, the cat's claw polypeptide functioned as a Delta 9 acyl-ACP desatu rase but displayed a distinct substrate specificity for palmitate (16: 0)-ACP rather than stearate (18:0)-ACP. Comparison of the predicted am ino acid sequence of the cat's claw enzyme with that of the castor Del ta 9-18:0-ACP desaturase suggested that a single amino acid substituti on (L118W) might account in large part for the differences in substrat e specificity between the two desaturases. Consistent with this predic tion, conversion of leucine-118 to tryptophan in the mature castor Del ta 9-18:0-ACP desaturase resulted in an 80-fold increase in the relati ve specificity of this enzyme for 16:0-ACP. The alteration in substrat e specificity observed in the L118W mutant is in agreement with a crys tallographic model of the proposed substrate-binding pocket of the cas tor Delta 9-18:0-ACP desaturase.