|Whittle, Edward - BROOKHAVEN NATIONAL LAB|
|Subrahmanyam, Satyam - BROOKHAVEN NATIONAL LAB|
|Shanklin, John - BROOKHAVEN NATIONAL LAB|
Submitted to: Journal of Biological Chemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: June 3, 2005
Publication Date: June 6, 2005
Citation: Whittle, E., Cahoon, E.B., Subrahmanyam, S., Shanklin, J. 2005. A multifunctional acyl-acyl carrier protein desaturase from hedera helix l. (english ivy) can synthesize 16- and 18-carbon monoene and diene products. Journal of Biological Chemistry. 280(31):28169-28176. Interpretive Summary: Fatty acid desaturases are enzymes that regulate the degree of unsaturation of vegetable oils and membranes. The activity of these enzymes is important for determining the nutritional quality and the potential industrial uses of vegetable oils. In this study, a novel fatty acid desaturase was identified from seeds of English ivy. The biochemical properties of this enzyme were determined, and a model was devised to describe how fatty acid substrates bind to the active site of this enzyme. It anticipated that this model will contribute to the rational design of fatty acid desaturases that can be used to produce new types of vegetable oils for nutritional and industrial applications. The findings of this study will be useful for biochemists and molecular biologists who are attempting to enhance the value of existing oilseed crops such as soybean. This research will ultimately expand the markets for oilseed crops grown by U.S. farmers and result in vegetable oils with greater utility for food scientists and industrial chemists.
Technical Abstract: A desaturase with 83% sequence identity to the coriander delta4-16:0-acyl carrier protein (ACP) desaturase was isolated from developing seeds of Hedera helix L. (English ivy). Expression of the ivy desaturase in Arabidopsis resulted in the accumulation of 16:1delta4 and its expected elongation product 18:1delta6 (petroselinic acid). The ivy desaturase was expressed in E. coli and purified to near homogeneity. In vitro assays confirmed the specificity of the desaturase to be delta4 with 16:0-ACP; however, with 18:0-ACP desaturation occurred at the delta9 position, implying two distinct substrate-binding modes, one for 16:0 placing C4 at the diiron active site, the other for 18:0 placing C9 at the active site. We also observed that 18:1delta9-ACP was further desaturated to 18:2delta4,9, and that 16:1delta9 was metabolized to 16:2delta4,9, both conversions occurring in a near-quantitative manner. Desaturation at the delta4 position requires that the substrate be inserted five carbons deeper into the substrate-binding cavity than for delta9 desaturation. Sequence comparison of the ivy and castor desaturases reveals a high degree of substrate binding-site conservation, but paradoxically, the specific substitutions identified, T117R and P179I (castor/ivy), are larger for smaller residues that decrease cavity size and were previously shown to favor binding fewer carbons beyond the diiron site. Substrate binding in the ivy desaturase can therefore not occur in the extended conformation described for the castor desaturase, and more likely occurs in a U-shaped conformation. Dual' delta4 and delta9 regiospecificity of the ivy desaturase, with 16C- and 18C-substrates respectively, makes it ideally suited for further dissection of the determinants of regiospecificity.