Objective
The flavoproteins ferredoxin nicotinamide adenine dinucleotide phosphate cation (NADP cation) reductase (FNR) and flavodoxin, function in photosynthesis and other low potential reactions. They are small proteins which may serve as models for larger flavoproteins, some of which (eg glucose oxidase, diaphorase) are already used widely in diagnostic kits and other biotechnological applications.
Knowledge of the factors which control the structure and function of FNR and flavodoxins could lead to the design of flavoenzymes suitable for biotechnological use. Molecular biology and protein engineering has been used to investigate the interactions between the 2 proteins and interactions between each flavoprotein and its flavin coenzyme.
An existing cloned gene of spinach FNR was expressed, to investigate the NADP cation binding site of the enzyme by site directed mutagenesis, and to study the interaction of this enzyme with flavodoxin from the sulphate reducing bacterium Desulfovibrio vulgaris by chemical crosslinking of the 2 proteins.
A complementary deoxyribonucleic acid (cDNA) clone for the preprotein of spinach FNR has been modified to allow expression in Escherichia coli of the nature flavoprotein in 2forms; a 35 kDa enzyme similar to that isolated from spinach and a 32 kDa enzyme with the same diaphorase activity but with impaired interaction with ferredoxin.
Site directed mutagenesis to produce spinach FNR-Lys116Gln and FNR-Lys244Gln and chemical modification studies with N-ethylmaleimide, combined with studies of the catalytic and substrate binding properties of the modified proteins, confirmed that both lysines are important in NADP(H) binding, that Lys116 has a main role in catalysis and that in contrast, the thiols of cysteines are not involved in catalysis.
Electrostatic and covalent 1:1 complexes of the FNRs and flavodoxins were made to investigate electron transfer between flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN) in proteins. The covalent complexes are catalytically active, and the FMN of flavodoxin is essential for electron transfer.
The redox potentials of FAD and FMN in the complexes of the 2 proteins differ from those in the free proteins, and the differences can be explained by changes on reduction of the strength of interaction of each of the 2 flavins with the protein (covalent complex) and/or of the interaction of the 2 proteins (electrostatic complex).
Clones of the genes for flavodoxins from Anabaena PCC 7119, a blue-green alga and Desulfovibrio vulgaris, a sulphate reducing bacterium, and for ferredoxin-nicotinamide adenine dinucleotide phosphate cation (NADP cation) reductases from spinach and Anabaena are now available; they have been sequenced and, with the exception of the reductase from Anabaena, all of the cloned genes have been expressed.
The first site-directed mutants of a flavodoxin and a ferredoxin NADP cation reductase have been prepared and characterized. These have provided information about the effects of protein structure on the substrate of flavin binding sites of the proteins.
New information has been obtained about the interaction between ferredoxin-NADP cation reductases and flavodoxins from structural studies and redox potentials of the two proteins following covalent linkage.
Fields of science (EuroSciVoc)
CORDIS classifies projects with EuroSciVoc, a multilingual taxonomy of fields of science, through a semi-automatic process based on NLP techniques. See: The European Science Vocabulary.
CORDIS classifies projects with EuroSciVoc, a multilingual taxonomy of fields of science, through a semi-automatic process based on NLP techniques. See: The European Science Vocabulary.
- natural sciences biological sciences microbiology bacteriology
- natural sciences biological sciences microbiology phycology
- natural sciences biological sciences genetics DNA
- natural sciences chemical sciences catalysis
- natural sciences biological sciences biochemistry biomolecules proteins enzymes
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Coordinator
20133 MILANO
Italy
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