title: 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.
abstract: Stearoyl-acyl carrier protein desaturase (Delta9D) 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-Delta9D 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-hydroxysuccinimide revealed that carboxylate residues from Fd and lysine residues from Delta9D contribute to cross-linking. The single substitutions of K60A, K56A, and K230A on Delta9D decreased the k(cat)/K(M) for Fd by 4-, 22-, and 2400-fold, respectively, as compared to wt Delta9D and a K41A substitution. The double substitution K56A/K60A decreased the k(cat)/K(M) for Fd by 250-fold, whereas the triple mutation K56A/K60A/K230A decreased the k(cat)/K(M) for Fd by at least 700 000-fold. These results strongly implicate the triad of K56, K60, and K230 of Delta9D 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 Delta9D and other protein-protein complexes.
authors.affiliation: Department of Biochemistry, College of Agricultural and Life Sciences, University of Wisconsin, Madison, Wisconsin 53706, USA.
authors.forename: Pablo
authors.initials: P
authors.lastname: Sobrado
authors.forename: Karen S
authors.initials: KS
authors.lastname: Lyle
authors.forename: Steven P
authors.initials: SP
authors.lastname: Kaul
authors.forename: Michelle M
authors.initials: MM
authors.lastname: Turco
authors.forename: Ida
authors.initials: I
authors.lastname: Arabshahi
authors.forename: Ashok
authors.initials: A
authors.lastname: Marwah
authors.forename: Brian G
authors.initials: BG
authors.lastname: Fox
mesh_terms: D001665:Binding Sites; D002384:Catalysis; D004579:Electron Transport; D005288:Ferredoxins / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D008024:Ligands; D008239:Lysine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006899:Mixed Function Oxygenases / Q000737:chemistry* / Q000378:metabolism; D008958:Models, Molecular; D016296:Mutagenesis; D010084:Oxidation-Reduction; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012264:Ribonucleotide Reductases / Q000737:chemistry / Q000378:metabolism; D014364:Tryptophan / Q000737:chemistry / Q000378:metabolism
chemical_list: C081714:2Fe-2S ferredoxin; D005288:Ferredoxins; D008024:Ligands; D014364:Tryptophan; D006899:Mixed Function Oxygenases; C018747:acyl-(acyl-carrier-protein)desaturase; D012264:Ribonucleotide Reductases; D008239:Lysine