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BIOENGINEERING OF FERREDOXIN NADP+ REDUCTASE AND FLAVODOXIN 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.

The genes for coding for flavodoxin and FNR in the blue green alga Anabaena 7199 have been clobed and expressed in E coli in high yields. Site directed mutagenesis of the two proteins allows the modification of the recognition behaviour. Modification of the residues in the surface of the enzyme (FNR) molecule allows the anchorage of the protein on the surface of a gold electrode for the exchange of electrons.

This stategy can be used for the recognition on NADP+ (NADPH) used in a biotransformation reaction catalysed by a second enzyme.

The electrochemically generated NADPH can be used by the enzyme cytochrome P450 to modify a steroid hormone (progesteraone of prenolone) to yield 17 alphahydroxy derivatives.
This same stategy could also be used to regenerate NADP+ in a reaction producing NADPH. The enzyme coated electrode could be used as a biosensor of NADP+ (NADPH)

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

UNIV. DE ZARAGOZA - DEPT. QUIMICA
Address
Ciudad Universitaria S/n
50009 Zaragoza
Spain