A single mutation in the castor 9-18:0-desaturase changes reaction partitioning from desaturation to oxidase chemistry. Xanya Sofra Weiss

A single mutation in the castor 9-18:0-desaturase changes reaction partitioning from desaturation to oxidase chemistry Jodie E. Guy*, Isabel A. Abreu†, Martin Moche‡, Ylva Lindqvist*, Edward Whittle†, and John Shanklin†§ *Department of Medical Biochemistry and Biophysics, Division of Molecular Structural Biology, Karolinska Institutet, Tomtebodava¨gen 6, S-171 77 Stockholm, Sweden; †Department of Biology, Brookhaven National Laboratory, Upton, NY 11973; and ‡Department of Medical Biochemistry and Biophysics and Structural Genomics Consortium, Karolinska Institutet, S-171 77 Stockholm, Sweden Edited by Christopher R. Somerville, Carnegie Institution of Washington, Stanford, CA, and approved September 28, 2006 (received for review August 17, 2006) 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 2 103-fold. However, it shows a >31-fold increase in peroxide-dependent oxidase activity with respect toWT desaturase, as monitored by single-turnover stopped-flow spectrometry. A 2.65-Å 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. binuclear diiron enzyme Nonheme diiron-containing four-helix-bundle proteins possess the ability to functionalize unactivated C-H groups and mediate a diversity of chemical reactions including oxidation, hydroxylation, desaturation, and epoxidation (1, 2). A wealth of mechanistic information is available from various diironcontaining proteins including methane monooxygenases, 9 desaturases, ribonucleotide reductases, rubrerythrins, alternate oxidases, ferritins, and bacterioferritins (1–3). The diiron-containing proteins are highly divergent in their amino acid sequences, with identities typically falling below that necessary for conventional phylogenetic analysis. However, when the analysis is restricted to the four helices that coordinate the diiron active site, the amino acid identity rises to 16–31% (4). A shared diiron-binding motif within the conserved four-helix bundle is involved in oxygen chemistry. The reactions have been described as occurring in two phases, an oxygen activation phase followed by reaction phases (1). Oxygen activation likely placed strong evolutionary constraints on the organization of the diiron center, whereas the reaction phases exhibit great diversity of functional outcome. In addition to their individual catalytic reactions, rubrerythrin, methane monooxygenase, ribonucleotide reductase, and the 9 desaturase have also been shown to reduce dioxygen to water (4–6). Based on these similarities, Gomes et al. (4) proposed that the four-helix bundle diiron proteins arose from a common ancestor that bound activated oxygen species and reduced them to water. This hypothetical oxidase enzyme is thought to have appeared at the transition from anaerobic to aerobic environment, 2.5 billion years ago. We previously performed a structural comparison of the active site of the 9 desaturase with that of rubrerythrin, an NAD(P)H peroxidase, which revealed remarkable similarity of the diiron ligands (7). Based on this structural analysis we proposed that residue 199, which occupies a location adjacent to the diiron site and abuts the hydrophobic substrate binding cavity, plays a key role in determining the chemical outcome of the enzyme (7). In the desaturase it is occupied by threonine, and in the rubrerythrin it is occupied by a glutamic acid. In this work we report that the T199D mutant of the 9 desaturase shows greatly reduced desaturation activity but increases its oxidase activity by 31-fold with respect to theWT desaturase. A crystal structure of the T199D mutant is presented that shows very close active-site similarity to rubrerythrin, consistent with its change in functionality. Results and Discussion Structural alignment of the reduced azide complexes of 9 desaturase and rubrerythrin (8) revealed similarities with respect to the position and identity of iron binding ligands and the position of the azide adduct (7) (Fig. 1). The single major difference in the active site is the identity of the residue corresponding to threonine-199 in the desaturase, which is a glutamic acid in rubrerythrin. The side chain of the residue occupying this position faces the bound azide that mimics the binding site of molecular oxygen. Thus, the desaturase contains threonine, a poor proton donor, whereas rubrerythrin contains a glutamic acid, which facilitates proton transfer. We previously hypothesized that the presence or absence of a proton donor in this position might influence the partitioning of chemical reactivity of the diiron site between desaturation and oxidase chemistry (7). Thus, we engineered mutations at position 199 into the desaturase to replace threonine with either glutamic or aspartic acid and compared the desaturase and oxidase activity of these mutants to those of WT 9 desaturase.

Xanya Sofra Weiss

Xanya Sofra Weiss