PDBsum entry 2j2f

Oxidoreductase

PDB id: 2j2f

Contents

Protein chains

(+ 0 more) 348 a.a.

Metals

_FE ×12

Waters ×158

References listed in PDB file

Key reference

Title

A single mutation in the castor delta9-18:0-Desaturase changes reaction partitioning from desaturation to oxidase chemistry.

Authors

J.E.Guy, I.A.Abreu, M.Moche, Y.Lindqvist, E.Whittle, J.Shanklin.

Ref.

Proc Natl Acad Sci U S A, 2006, 103, 17220-17224. [DOI no: 10.1073/pnas.0607165103]

PubMed id

17088542

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.

Figure 3.
Fig. 3. A schematic to describe the reaction of the desaturase T199D.

Figure 4.
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.