A desaturase with 83% sequence identity to the coriander ⌬ -ACP to the corresponding ⌬ 4,9 dienes. These data suggest at least two distinct substrate binding modes, one placing C4 at the diiron active site and the other placing C9 at the active site. In the latter case, 18:0 would likely bind in an extended conformation as described for the castor desaturase with 9-carbons accommodated in the cavity beyond the dirron site. However, ⌬ 4 desaturation would require the accommodation of 12 carbons for C16 substrates or 14 carbons for C18 substrates. The amino acids lining the substrate binding cavity of ivy and castor desaturases are conserved except for T117R and P179I (castor/ivy). Paradoxically, both substitutions, when introduced into the castor desaturase, favored the binding of shorter acyl chains. Thus, it seems likely that ⌬ 4 desaturation would require a non-extended, perhaps U-shaped, substrate conformation. A cis double bond may facilitate the initiation of such a non-extended conformation in the monounsaturated substrates. The multifunctional properties of the ivy desaturase make it well suited for further dissection of the determinants of regiospecificity. Acyl-acyl carrier protein (ACP)1 desaturases (EC 1.14.99.6) are responsible for the conversion of saturated fatty acyl-ACPs into monounsaturated-ACPs (1-3). This reaction is the penultimate step in the ACP track of fatty acid biosynthesis in the plastid and represents the major source of monounsaturated fatty acids in plant tissues (4). The resulting monounsaturated fatty acids are required to maintain membrane fluidity and in storage tissues are directed into storage triacylglycerols either directly or after additional desaturation steps. Plant acyl-ACP desaturases belong to a distinct sequence class composed of soluble globular proteins, whereas most fatty acid desaturases, found in fungi and animal and plants, are integral membrane proteins (5). All desaturases identified to date that recognize monoene, diene, and polyene substrates are members of the integral membrane class of desaturases. The archetypal ⌬ 9 -18: 0-ACP 2 desaturase from Ricinus communis (castor) has been the focus of extensive structure-function studies because it is readily expressed in active form in Escherichia coli, and it is the only member of the acyl-ACP desaturase family for which a three-dimensional crystal structure has been determined (6, 7). Each monomer of the castor enzyme homodimer contains a boomerang-shaped cavity capable of binding a stearoyl moiety in a gauche conformation adjacent to the active site diiron cluster. Although a crystal structure of the desaturase-substrate complex has yet to be published, modeling the substrate into the existing crystal structure suggests that when the methyl group of the stearate is in contact with the bottom of the cavity, C9 and C10 are positioned with their pro-R hydrogens facing the diiron active site (6). This structural model is consistent with the Pro-R Pro-R stereochemistry reported for the desaturase reaction (8) and th...