PDBsum entry 2j2f

Oxidoreductase

PDB id: 2j2f

Contents

Protein chains

(+ 0 more) 348 a.a. *

Metals

_FE ×12

Waters ×158

* Residue conservation analysis

PDB id:

2j2f

Name:

Oxidoreductase

Title:

The t199d mutant of stearoyl acyl carrier protein desaturase from ricinus communis (castor bean)

Structure:

Acyl-[acyl-carrier-protein] desaturase. Chain: a, b, c, d, e, f. Fragment: residues 34-396. Synonym: stearoyl acp desaturase, delta 9 stearoyl-acyl carrier protein desaturase. Engineered: yes. Mutation: yes

Source:

Ricinus communis. Castor bean. Organism_taxid: 3988. Expressed in: escherichia coli. Expression_system_taxid: 469008.

Biol. unit:

Dimer (from PDB file)

Resolution:

2.65Å R-factor: 0.242 R-free: 0.272

Authors:

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

Key ref:

J.E.Guy et al. (2006). A single mutation in the castor Delta9-18:0-desaturase changes reaction partitioning from desaturation to oxidase chemistry. Proc Natl Acad Sci U S A, 103, 17220-17224. PubMed id: 17088542 DOI: 10.1073/pnas.0607165103

Date:

16-Aug-06 Release date: 23-Oct-06

Protein chains

P22337  (STAD_RICCO) -  Stearoyl-[acyl-carrier-protein] 9-desaturase, chloroplastic from Ricinus communis

Seq: 396 a.a.

Struc: 348 a.a.*

* PDB and UniProt seqs differ at 1 residue position (black cross)

Enzyme reactions

• Enzyme class: E.C.1.14.19.2 - stearoyl-[acyl-carrier-protein] 9-desaturase.
• Reaction: octadecanoyl-[ACP] + 2 reduced [2Fe-2S]-[ferredoxin] + O2 + 2 H⁺ = (9Z)-octadecenoyl-[ACP] + 2 oxidized [2Fe-2S]-[ferredoxin] + 2 H2O

Molecule diagrams generated from .mol files obtained from the KEGG ftp site

Key reference

DOI no: 10.1073/pnas.0607165103

Proc Natl Acad Sci U S A 103:17220-17224 (2006)

PubMed id: 17088542

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

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

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.

Selected figure(s)

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.

Figures were selected by an automated process.