The Crystal Structure of the Ivy Δ4-16:0-ACP Desaturase Reveals Structural Details of the Oxidized Active Site and Potential Determinants of Regioselectivity

Abstract

The multifunctional acyl-acyl carrier protein (ACP) desaturase from Hedera helix (English ivy) catalyzes the ⌬ 4 desaturation of 16:0-ACP and the ⌬ 9 desaturation of 18:0-ACP and further desaturates ⌬ 9 -16:1 or ⌬ 9 -18:1 to the corresponding ⌬ 4,9 dienes. The crystal structure of the enzyme has been solved to 1.95 Å resolution, and both the iron-iron distance of ϳ3.2 Å and the presence of a -oxo bridge reveal this to be the only reported structure of a desaturase in the oxidized FeIII-FeIII form. Significant differences are seen between the oxidized active site and the reduced active site of the Ricinus communis (castor) desaturase; His 227 coordination to Fe2 is lost, and the side chain of Glu 224 , which bridges the two iron ions in the reduced structure, does not interact with either iron. Although carboxylate shifts have been observed on oxidation of other diiron proteins, this is the first example of the residue moving beyond the coordination range of both iron ions. Comparison of the ivy and castor structures reveal surface amino acids close to the annulus of the substrate-binding cavity and others lining the lower portion of the cavity that are potential determinants of their distinct substrate specificities. We propose a hypothesis that differences in side chain packing explains the apparent paradox that several residues lining the lower portion of the cavity in the ivy desaturase are bulkier than their equivalents in the castor enzyme despite the necessity for the ivy enzyme to accommodate three more carbons beyond the diiron site. Fatty acid acyl carrier protein (ACP)3 desaturases (EC 1.14.99.6) convert saturated fatty acyl-ACPs into their cis-monounsaturated equivalents in an oxygen-dependent reaction (1-6). ⌬ 9 -desaturation of 18:0-ACP 4 occurs ubiquitously in the plastids of plants and thus plays an important role in determining the fluidity of cell membranes (7). Plants can express distinct acyl ACP desaturases, e.g. Arabidopsis thaliana has a family of seven acyl-ACP desaturases that are differentially expressed in different tissues (8). A structurally related homolog with unknown function from Mycobacterium has also been described (9).Seed oils of various plants accumulate monounsaturated fatty acids with unusual chain lengths and/or double bond positions. Several ⌬ 9 desaturases that recognize 14-, 16-, and 18-carbon chain lengths (4, 10 -12) have been described, and others with ⌬ 6 and ⌬ 9 regiospecificities have also been reported (13,14). The biosynthetic origin of unusual fatty acids is not always straightforward; for instance, Coriandrum sativum L. (coriander) and Hedera helix (English ivy) accumulate petroselinic acid (18:1⌬ 6 ) that originates via a ⌬ 4 desaturation of 16:0-ACP followed by 2-carbon elongation to 18:1⌬ 6 (15). The genes encoding these ⌬ 4 -16:0-ACP desaturase enzymes have been isolated (15,16), and their amino acid sequences are homologous to that of the castor ⌬ 9 -18:0-ACP desaturase for which the crystal structure has been determined (17,18). This implies that ac...

FAD2 and FAD3 Desaturases Form Heterodimers That Facilitate Metabolic Channeling in Vivo

“…The proportions of endogenous unlabeled, d2, or 13 C-labeled fatty acid in PC are presented in Table 2. Although both labeled precursors were efficiently incorporated into PC-18:3⌬ 9,12,15 , the label distribution in PC-bound 18:2 and 18:3 differs considerably. However, if all PC-bound 18:3 acyl chains had been derived from 18:2 acyl chains bound to the same PC pool, the label distribution should have been equivalent.…”

“…However, if all PC-bound 18:3 acyl chains had been derived from 18:2 acyl chains bound to the same PC pool, the label distribution should have been equivalent. As described above, the conversion of PC-bound 18:1⌬ 9 to 18:3⌬ 9,12,15 can take place by two distinct routes, either via desaturation by discrete FAD2 and FAD3 homodimers or via sequential coupled desaturation by FAD2-FAD3 heterodimers (Fig. 8).…”

“…For instance the crystal structure of the castor and ivy acyl-ACP desaturases confirmed their homodimeric organization mediated by deep interdigitation of loops between protomers adjacent to the four-helix bundles in which the diiron active site is located (14,15). Based on the dimeric structure of soluble desaturase, a mechanism involving half-of-the-sites reactivity was proposed in which the desolvation energy released upon substrate binding would be transferred to the second subunit to help facilitate product release (16), the proposed limiting step in desaturation (17).…”

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

“…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.…”

A Small Membrane-peripheral Region Close to the Active Center Determines Regioselectivity of Membrane-bound Fatty Acid Desaturases from Aspergillus nidulans

“…One conceivable model how changes in regiospecificity may have developed in evolution is by gene duplication and random accumulation of mutations in the catalytic portion of the enzymes to produce first a broader and then a different specificity (10,12). Changes to an enzyme regiospecificity typically require between two and six specific alterations at key locations along the amino acid chain (6,14). According to the described model the first step toward a new specificity of an enzyme is the accumulation of mutations in the corresponding gene, which result in decreased enzyme specificity and lead to broader substrate acceptance and/or formation of multiple products (12).…”