title: 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.
authors.affiliation: Department of Medical Biochemistry and Biophysics, Division of Molecular Structural Biology, Karolinska Institutet, Tomtebodavägen 6, S-171 77 Stockholm, Sweden.
authors.forename: Jodie E
authors.initials: JE
authors.lastname: Guy
authors.forename: Isabel A
authors.initials: IA
authors.lastname: Abreu
authors.forename: Martin
authors.initials: M
authors.lastname: Moche
authors.forename: Ylva
authors.initials: Y
authors.lastname: Lindqvist
authors.forename: Edward
authors.initials: E
authors.lastname: Whittle
authors.forename: John
authors.initials: J
authors.lastname: Shanklin
mesh_terms: D001665:Binding Sites; D018360:Crystallography, X-Ray; D044943:Fatty Acid Desaturases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics; D010088:Oxidoreductases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D013230:Stearoyl-CoA Desaturase
chemical_list: D010088:Oxidoreductases; D044943:Fatty Acid Desaturases; D013230:Stearoyl-CoA Desaturase; C029627:delta-9 fatty acid desaturase