Swapping FAD binding motifs between plastidic and bacterial ferredoxin-NADP(H) reductases
| Autor: | Alejandro Buschiazzo, Horacio Botti, Eduardo A. Ceccarelli, Matías A. Musumeci |
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| Přispěvatelé: | Instituto de Biología Molecular y Celular de Rosario [Rosario] (IBR), Consejo Nacional de Investigaciones Científicas y Técnicas [Buenos Aires] (CONICET)-Universidad Nacional de Rosario [Santa Fe], Institut Pasteur de Montevideo, Réseau International des Instituts Pasteur (RIIP), Biochimie Structurale, Centre National de la Recherche Scientifique (CNRS)-Institut Pasteur [Paris], This work was supported by grants from CONICET and the Agencia de Promocion Científica y Tecnologica (ANPCyT), Argentina. E.A.C. is a staff member of the Consejo Nacional de Investigaciones Científicas y Tecnicas (CONICET, Argentina). M.A.M. is a fellow of the same institution. Financial and logistic support from the Institut Pasteur de Montevideo (Protein Crystallography Facility PXF), the Amsud/Pasteur network, and the Center of Structural Biology of the Mercosur (CeBEM) is gratefully acknowledged., Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS) |
| Jazyk: | angličtina |
| Rok vydání: | 2011 |
| Předmět: |
Models
Molecular 0106 biological sciences MESH: Enzyme Stability Amino Acid Motifs MESH: Amino Acid Sequence Crystallography X-Ray environment and public health 01 natural sciences Biochemistry MESH: Amino Acid Motifs Diaphorase Enzyme Stability Plastids Ferredoxin chemistry.chemical_classification 0303 health sciences MESH: Kinetics MESH: Escherichia coli Temperature MESH: Plastids MESH: Temperature Ferredoxin-NADP Reductase MESH: Protein Unfolding MESH: Flavin-Adenine Dinucleotide Flavin-Adenine Dinucleotide MESH: Models Molecular inorganic chemicals Molecular Sequence Data Cytochrome c reductase Biology MESH: Peas 03 medical and health sciences Oxidoreductase Escherichia coli [SDV.BBM]Life Sciences [q-bio]/Biochemistry Molecular Biology Amino Acid Sequence Enzyme kinetics Protein Unfolding 030304 developmental biology MESH: Molecular Sequence Data fungi Peas Tryptophan MESH: Crystallography X-Ray Kinetics enzymes and coenzymes (carbohydrates) Enzyme chemistry FAD binding bacteria MESH: Ferredoxin-NADP Reductase 010606 plant biology & botany |
| Zdroj: | Biochemistry Biochemistry, American Chemical Society, 2011, 50 (12), pp.2111-22. ⟨10.1021/bi101772a⟩ Biochemistry, 2011, 50 (12), pp.2111-22. ⟨10.1021/bi101772a⟩ |
| ISSN: | 0006-2960 1520-4995 |
| DOI: | 10.1021/bi101772a⟩ |
| Popis: | International audience; Plant-type ferredoxin-NADP(H) reductases (FNRs) are grouped in two classes, plastidic with an extended FAD conformation and high catalytic rates and bacterial with a folded flavin nucleotide and low turnover rates. The 112-123 β-hairpin from a plastidic FNR and the carboxy-terminal tryptophan of a bacterial FNR, suggested to be responsible for the FAD differential conformation, were mutually exchanged. The plastidic FNR lacking the β-hairpin was unable to fold properly. An extra tryptophan at the carboxy terminus, emulating the bacterial FNR, resulted in an enzyme with decreased affinity for FAD and reduced diaphorase and ferredoxin-dependent cytochrome c reductase activities. The insertion of the β-hairpin into the corresponding position of the bacterial FNR increased FAD affinity but did not affect its catalytic properties. The same insertion with simultaneous deletion of the carboxy-terminal tryptophan produced a bacterial chimera emulating the plastidic architecture with an increased k(cat) and an increased catalytic efficiency for the diaphorase activity and a decrease in the enzyme's ability to react with its substrates ferredoxin and flavodoxin. Crystallographic structures of the chimeras showed no significant changes in their overall structure, although alterations in the FAD conformations were observed. Plastidic and bacterial FNRs thus reveal differential effects of key structural elements. While the 112-123 β-hairpin modulates the catalytic efficiency of plastidic FNR, it seems not to affect the bacterial FNR behavior, which instead can be improved by the loss of the C-terminal tryptophan. This report highlights the role of the FAD moiety conformation and the structural determinants involved in stabilizing it, ultimately modulating the functional output of FNRs. |
| Databáze: | OpenAIRE |
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