A single mutation in the castor Delta9-18:0-desaturase changes reaction partitioning from desaturation to oxidase chemistry

Abstract

Sequence analysis of the diiron cluster-containing soluble desaturases suggests they are unrelated to other diiron enzymes; however, structural alignment of the core four-helix bundle of desaturases to other diiron enzymes reveals a conserved iron binding motif with similar spacing in all enzymes of this structural class, implying a common evolutionary ancestry. Detailed structural comparison of the castor desaturase with that of a peroxidase, rubrerythrin, shows remarkable conservation of both identity and geometry of residues surrounding the diiron center, with the exception of residue 199. Position 199 is occupied by a threonine in the castor desaturase, but the equivalent position in rubrerythrin contains a glutamic acid. We previously hypothesized that a carboxylate in this location facilitates oxidase chemistry in rubrerythrin by the close apposition of a residue capable of facilitating proton transfer to the activated oxygen (in a hydrophobic cavity adjacent to the diiron center based on the crystal structure of the oxygen-binding mimic azide). Here we report that desaturase mutant T199D binds substrate but its desaturase activity decreases by approximately 2 x 10(3)-fold. However, it shows a >31-fold increase in peroxide-dependent oxidase activity with respect to WT desaturase, as monitored by single-turnover stopped-flow spectrometry. A 2.65-A crystal structure of T199D reveals active-site geometry remarkably similar to that of rubrerythrin, consistent with its enhanced function as an oxidase enzyme. That a single amino acid substitution can switch reactivity from desaturation to oxidation provides experimental support for the hypothesis that the desaturase evolved from an ancestral oxidase enzyme.

Fig. 1.

Crystal structures of the reduced azide complexes of desaturase (Upper) and rubrerythrin (Lower).

Fig. 2.

Pseudo first-order rate constants and H₂O₂ concentration dependency for the WT (●), T199E (○), and T199D (○) mutant proteins determined at 10°C.

Fig. 3.

A schematic to describe the reaction of the desaturase T199D.

Fig. 4.

A view of the superimposed active sites of the desaturase T199D mutant (green) and of reduced rubrerythrin (blue), showing the similar position of the putative proton donor groups.

Fig. 5.

The active site of the T199D mutant, showing an omit map (contoured at 3σ) of the difference density that was ultimately modeled as two water molecules.