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
Stearoyl-acyl carrier protein desaturase (Δ9D) catalyzes the O2 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...
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| Published in: | Biochemistry (Easton) Vol. 45; no. 15; pp. 4848 - 4858 |
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| Main Authors: | , , , , , , |
| Format: | Journal Article |
| Language: | English |
| Published: |
United States
American Chemical Society
18-04-2006
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| Subjects: | |
| Online Access: | Get full text |
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| Summary: | Stearoyl-acyl carrier protein desaturase (Δ9D) catalyzes the O2 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-hydroxysuccinimide 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/K M 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 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 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. |
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| Bibliography: | This work was supported by the National Institutes of Health Grant GM-50853 to B.G.F. P.S. was supported in part by a postdoctoral fellowship from CONICIT-Costa Rica (Grant 295-2004). K.S.L. was a trainee of the NIH Institutional Molecular Biophysics Pre-Doctoral Training Grant T32 GM-08293. istex:0185A39B7F2D0A1995A1B85561BEE156D8FCCEA9 ark:/67375/TPS-9ZVWK31T-B P.S. was supported in part by a post-doctoral fellowship from CONICIT-Costa Rica (Grant 295-2004). These two authors contributed equally to completion of the work. To whom correspondence should be addressed. Email: bgfox@biochem.wisc.edu. Telephone: (608) 262-9708. Fax: (608) 265-2904. K.S.L was a trainee of the NIH Institutional Molecular Biophysics Pre-Doctoral Training Grant T32 GM-08293. Zornetzer. G.A., Fox, B.G. and Markley, J.L., unpublished results. |
| ISSN: | 0006-2960 1520-4995 |
| DOI: | 10.1021/bi0600547 |