Abstract
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.
Highlights
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 [1,2,3,4,5,6]. ⌬9-desaturation of 18:0-ACP4 occurs ubiquitously in the plastids of plants and plays an important role in determining the fluidity of cell membranes [7]
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 acyl-ACP desaturases share a common architecture that is able to accommodate different substrate binding modes, which leads to the observed desaturation of fatty acids of different chain lengths and/or at different position along the fatty acid
The study of chimeras of the ϳ360-amino acid sequences of the mature castor ⌬9-18:0- and the Thunbergia ⌬6-16:0-ACP desaturases and information from the crystal structure of the castor ⌬9 –18:0-ACP desaturase led to the identification of five residues in the castor sequence that, when substituted into the corresponding positions in the Thunbergia sequence, con
Summary
Verted the Thunbergia ⌬6-16:0-ACP desaturase into ⌬9-18:0ACP desaturase [19]. subsequent attempts to resolve which residues contribute to chain length specificity and which to regioselectivity were unsuccessful. Modeling the substrate in an extended conformation into the cavity suggests that when the methyl group of the stearate is in contact with the bottom of the cavity, C-9 and C-10 are positioned with their pro-R hydrogens facing the diiron active site consistent with the introduction of a cis ⌬9 double bond [17] and the Pro-R, Pro-R, stereochemistry reported for castor desaturation [21]. This model is consistent with the location of the azide in the desaturase-azide complex, which mimics the active site oxidant responsible for hydrogen abstraction [18]. Comparison of this structure with that of the reduced castor desaturase reveals that the side chain of Glu224, which bridges the two iron ions in the reduced state, undergoes a large carboxylate shift upon oxidation, completely disengaging from the iron ions
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