A Multifunctional Acyl-Acyl Carrier Protein Desaturase from Hedera helix L. (English Ivy) Can Synthesize 16- and 18-Carbon Monoene and Diene Products

Abstract

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...

Remote control of regioselectivity in acyl-acyl carrier protein-desaturases

“…In contrast, the ivy desaturase exhibits exclusively Δ4 selectivity with 14 and 16 carbon substrates but, surprisingly, with 18 carbon substrate displays almost exclusively (97%) Δ9 desaturation. We previously solved the crystal structures of these two desaturases, the Δ9-18∶0-ACP desaturase from castor (4) and the Δ4-16∶0-ACP desaturase from ivy (8). The two structures were very similar, as predicted by the 76% amino acid sequence identity, and a detailed comparison yielded few insights into the basis for their different regioselectivities, which involves a shift in register of five carbons on the acyl chain with respect to the diiron active site.…”

Identification of bifunctional Δ12/ω3 fatty acid desaturases for improving the ratio of ω3 to ω6 fatty acids in microbes and plants

“…Although it has previously been shown that desaturases can have additional functionality, such as acting as conjugases (25) or hydroxylases (26), different positional specificities depending on the type of lipid to which the substrate is conjugated (27), the chain length of the acyl group (28), or the ability of a soluble desaturase enzyme to catalyze two double bonds (⌬4 and ⌬9) into a saturated fatty acyl-ACP substrate (29), the ability of a desaturase enzyme to catalyze two double bonds (⌬12 and 3) on the same acyl chain (C18) has not previously been reported. It will be interesting to study how these ⌬12͞ 3 enzymes exert their dual positional specificity, because the regioselectivity of ⌬12 and 3 desaturases is Јv ϩ 3Ј (i.e., three carbons away from an existing double bond) and Ϫ3 (i.e., three carbons from the methyl end), respectively (30,31).…”

Desaturases: Emerging Models for Understanding Functional Diversification of Diiron-containing Enzymes

“…They vary in both chain length selectivity and regioselectivity (35,36). A comparison of the structures of the castor 18:0 ⌬ 9 -and ivy 16:0 ⌬ 4 -desaturases (14) was initiated to determine the basis for regioselectivity (15). A triple mutant designed to investigate the basis for regioselectivity retained its ⌬ 9 -desaturation activity, converting 18:0 into 18:1⌬ 9-cis ; however, this product was further metabolized to 9-OH 18:1⌬ 10-trans (37) in a reaction reminiscent of the synthesis of dimorphecolic acid (9-OH 18: 2⌬ 10-trans,12-trans ) (38).…”

“…The availability of crystal structures for acyl-ACP desaturases (12) makes this system amenable to detailed structure-function studies. Crystal structures are available for the 18:0 ⌬ 9 -desaturase 3 (12,13) from Ricinus communis (castor) and a bifunctional desaturase from Hedera helix (ivy) (14,15). These desaturases are homodimeric proteins, with each monomer folded into a compact single domain composed of nine helices.…”