Abstract
Stearoyl-acyl carrier protein desaturase (Δ9D) catalyzes the O 2 and 2e- dependent desaturation of stearoyl-acyl carrier protein (18:0-ACP) to yield oleoyl-ACP (18:1-ACP). The 2e- are provided by essential interactions with reduced plant-type [2Fe-2S] ferredoxin (Fd). We have investigated the protein-protein interface involved in the Fd-Δ9D complex by the use of chemical cross-linking, site-directed mutagenesis, steady-state kinetic approaches, and molecular docking studies. The treatment of the different proteins with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and N-hydroxy succinimide revealed that carboxylate residues from Fd and lysine residues from Δ9D contribute to cross-linking. The single substitutions of K60A, K56A, and K230A on Δ9D decreased the k cat/KM for Fd by 4-, 22-, and 2400-fold, respectively, as compared to wt Δ9D and a K41A substitution. The double substitution K56A/K60A decreased the kcat/KM for Fd by 250-fold, whereas the triple mutation K56A/K60A/K230A decreased the kcat/K M for Fd by at least 700 000-fold. These results strongly implicate the triad of K56, K60, and K230 of Δ9D in the formation of a catalytic complex with Fd. Molecular docking studies indicate that electrostatic interactions between K56 and K60 and the carboxylate groups on Fd may situate the [2Fe-2S] cluster of Fd closer to W62, a surface residue that is structurally conserved in both ribonucleotide reductase and mycobacterial putative acyl-ACP desaturase DesA2. Owing to the considerably larger effects on catalysis, K230 appears to have other contributions to catalysis arising from its positioning in helix 7 and its close spatial location to the diiron center ligands E229 and H232. These results are considered in the light of the presently available models for Fd-mediated electron transfer in Δ9D and other protein-protein complexes. © 2006 American Chemical Society.
Recommended Citation
P. Sobrado et al., "Identification of the Binding Region of the [2Fe-2S] Ferredoxin in Stearoyl-acyl Carrier Protein Desaturase: Insight into the Catalytic Complex and Mechanism of Action," Biochemistry, vol. 45, no. 15, pp. 4848 - 4858, American Chemical Society, Apr 2006.
The definitive version is available at https://doi.org/10.1021/bi0600547
Department(s)
Chemistry
International Standard Serial Number (ISSN)
0006-2960
Document Type
Article - Journal
Document Version
Citation
File Type
text
Language(s)
English
Rights
© 2024 American Chemical Society, All rights reserved.
Publication Date
18 Apr 2006
PubMed ID
16605252
Comments
National Institute of General Medical Sciences, Grant T32GM008293