2J2F

The T199D Mutant of Stearoyl Acyl Carrier Protein Desaturase from Ricinus Communis (Castor Bean)

Summary for 2J2F

• Entry DOI: 10.2210/pdb2j2f/pdb
• Related: 1AFR 1OQ4 1OQ7 1OQ9 1OQB
• Descriptor: ACYL-[ACYL-CARRIER-PROTEIN] DESATURASE, FE (III) ION (3 entities in total)
• Functional Keywords: electron transfer, four-helix bundle, oxidoreductase, lipid synthesis, transit peptide, fatty acid biosynthesis, nadp, chloroplast, di-ron enzyme
• Biological source: RICINUS COMMUNIS (CASTOR BEAN)
• Cellular location: Plastid, chloroplast: P22337
• Total number of polymer chains: 6
• Total formula weight: 250973.92

Authors

Guy, J.E.,Abreu, I.A.,Moche, M.,Lindqvist, Y.,Whittle, E.,Shanklin, J. (deposition date: 2006-08-16, release date: 2006-10-23, Last modification date: 2023-12-13)

Primary citation

Guy, J.E.,Abreu, I.A.,Moche, M.,Lindqvist, Y.,Whittle, E.,Shanklin, J.
A Single Mutation in the Castor {Delta}9-18:0- Desaturase Changes Reaction Partitioning from Desaturation to Oxidase Chemistry.
Proc.Natl.Acad.Sci.USA, 103:17220-, 2006

PubMed 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.

PubMed: 17088542
DOI: 10.1073/PNAS.0607165103

Experimental method

X-RAY DIFFRACTION (2.65 Å)