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BIOENGINEERING OF FERREDOXIN - NADP REDUCTASE AND FLAVODIN; DIAGNOSTIC AND INDUSTRIAL APPLICATIONS

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.

Site directed mutagenesis and chemical modification have been used to assess the importance of specific amino acids in maintaining the protein structure and redox properties of the flavin mononucleotide (FMN) in flavodoxin from Desulfovibrio vulgaris. Expression of an existing gene of D. vulgaris flavodoxin in Escherichia coli was improved 400-fold. Site directed mutagenesis to produce D. vulgaris flavodoxin-Gly61Asn showed that the FMN semiquinone is less stable in the mutant, and the changes in redox potential and FMN binding constants suggested that the bulky sidechain of the asparagine hinders a conformation change and hydrogen bond formation that has been proposed between nitrogen(5)hydrogen of the flavin semiquinone and the backbone carbonyl of residue 61. Additional mutants of spinach FNR and D. vulgaris flavodoxin have been produced and are being characterised biochemically.

Electron transfer between flavin adenine dinuclestide (FAD) and FMN in the proteins was investigated. 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 c omplex) 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.

Funding Scheme

CSC - Cost-sharing contracts

Coordinator

UNIVERSITY COLLEGE DUBLIN
Address
Belfield Dublin 4

Ireland