Reduction of the Pea Ferredoxin-nadp(H) Reductase Catalytic Efficiency by the Structuring of a Carboxyl-terminal Artificial Metal Binding Site.
From: Molecular Biology Division, Instituto de Biología Molecular y Celular de Rosario, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Suipacha 531, S2002LRK Rosario, Argentina.
Biochemistry
- Publish Date: Nov 2006
- ISSN: 0006-2960
- Volume: 45
- Issue: 46
- Pages: 13899-909
- Medium: Print
- Language: English
- Citation (JAMA): Catalano-Dupuy Daniela L, Orecchia Martín, Rial Daniela V, et al. Reduction of the Pea Ferredoxin-nadp(H) Reductase Catalytic Efficiency by the Structuring of a Carboxyl-terminal Artificial Metal Binding Site.. Biochemistry Nov 2006;45:13899-909
Abstract
Ferredoxin (flavodoxin)-NADP(H) reductases (FNRs) are ubiquitous flavoenzymes that deliver NADPH or low-potential one-electron donors (ferredoxin, flavodoxin, and adrenodoxin) to redox-based metabolisms in plastids, mitochondria, and bacteria. The FNRs from plants and most eubacteria constitute a unique family, the plant-type ferredoxin-NADP(H) reductases. Plastidic FNRs are quite efficient at sustaining the demands of the photosynthetic process. At variance, FNRs from organisms with heterotrophic metabolisms or anoxygenic photosynthesis display turnover numbers that are 20-100-fold lower than those of their plastidic and cyanobacterial counterparts. To gain insight into the FNR structural features that modulate enzyme catalytic efficiency, we constructed a recombinant FNR in which the carboxyl-terminal amino acid (Tyr308) is followed by an artificial metal binding site of nine amino acids, including four histidine residues. This added structure binds Zn2+ or Co2+ and, as a consequence, significantly reduces the catalytic efficiency of the enzyme by decreasing its kcat. The Km for NADPH and the Kd for NADP+ were increased 2 and 3 times, respectively, by the addition of the amino acid extension in the absence of Zn2+. Nevertheless, the structuring of the metal binding site did not change the Km for NADPH or the Kd for NADP+ of the FNR-tail enzyme. Our results provide experimental evidence which indicates that mobility of the carboxyl-terminal backbone region of the FNR, mainly Tyr308, is essential for obtaining an FNR enzyme with high catalytic efficiency.
Mesh Headings (Keywords): Amino Acid Sequence, Base Sequence, Binding Sites, Catalysis, DNA, Plant, Ferredoxin-NADP Reductase, Kinetics, Metals, Peas, Recombinant Proteins, Spectrometry, Fluorescence, X-Ray Diffraction
Check for Full Text / PubMed Unique Identifier (PMID): 17105208
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