Desaturases: emerging models for understanding functional diversification of diiron-containing enzymes

Abstract

Desaturases and related enzymes perform O(2)-dependent dehydrogenations initiated at unactivated C-H groups with the use of a diiron active site. Determination of the long-sought oxidized desaturase crystal structure facilitated structural comparison of the active sites of disparate diiron enzymes. Experiments on the castor desaturase are discussed that provide experimental support for a hypothesized ancestral oxidase enzyme in the context of the evolution of the diiron enzyme diverse functionality. We also summarize recent analysis of a castor mutant desaturase that provides valuable insights into the relationship of proposed substrate-binding modes with respect to a range of catalytic outcomes.

FIGURE 1.

Superimposition of the reduced active site of the castor desaturase and the oxidized active site of the ivy desaturase. A, the reduced active site is shown in blue gray, with brown irons and gray water (Wat). The oxidized active site is shown in cyan, with orange iron ions, and the μ-oxo bridge and water are depicted in yellow. B, the active-site residues of the azide complex of the castor desaturase are shown, with colors the same as those described for A and with Thr¹⁹⁹ included. The Asp¹⁹⁹ side chain of the T199D mutant is superimposed on the structure and is depicted in green. Note that the view is from the opposite face of the active site with respect to A to clearly show the position of the mutation.

FIGURE 2.

Schematic representation of the proposed binding modes of substrate (inner) and their respective products (outer). Note the substrate enters the channel from the right-hand side in the extended conformation.