The crystal structure of the ivy Delta4-16:0-ACP desaturase reveals structural details of the oxidized active site and potential determinants of regioselectivity.

The multifunctional acyl-acyl carrier protein (ACP) desaturase from Hedera helix (English ivy) catalyzes the Delta(4) desaturation of 16:0-ACP and the Delta(9) desaturation of 18:0-ACP and further desaturates Delta(9)-16:1 or Delta(9)-18:1 to the corresponding Delta(4,9) dienes. The crystal structure of the enzyme has been solved to 1.95 A resolution, and both the iron-iron distance of approximately 3.2A and the presence of a mu-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.