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Antoine Picciocchi

Functional characterization of biotin synthase reaction in Arabidopsis thaliana: The importance of plant mitochondria in biotin synthesis

Published on 16 December 2003

Thesis presented December 16, 2003

Biotin (vitamin H or B8) is exclusively synthesized by plants and microorganisms. In plant cells, the last step of the biotin biosynthetic pathway is catalyzed by the biotin synthase protein. An Arabidopsis thaliana cDNA, called bio2, had been previously isolated in the laboratory, and the corresponding protein over-expressed in Escherichia coli cells and purified. However, no biotin synthase activity was associated with this protein, so that the mechanism of the plant enzyme reaction was still unknown.
The development of biochemical sensitive tests allowed the first detection and accurate quantification of a plant biotin synthase activity, using protein extracts from bacteria overexpressing the Arabidopsis Bio2 protein. Under optimized assay conditions, the turnover number of the reaction was > 2h-1, indicating that biotin synthase from Arabidopsis is not, as suggested for bacteria, a simple reactant. The purification of the A. thaliana recombinant protein shows that bio2 gene product, like its bacterial counterpart, needs additional proteins to function. In an in vitro plant-reconstituted system, only mitochondrial fractions (prepared from pea leaves and potato tubers) efficiently restore the biotin synthase activity driven by Bio2 protein. A biochemical screening of potato mitochondrial matrix (fractionation and in vitro reconstitution experiments) together with a genomic based research in the A. thaliana databank allowed us to identify mitochondrial adrenodoxin (Adx), adrenodoxin reductase (AdxR) and cysteine desulfurase (Nfs1) proteins as essential components for the plant biotin synthase reaction. Arabidopsis cDNAs encoding these proteins were cloned into expression vectors, and the corresponding proteins overexpressed in E. coli cells. The in vitro stimulation of biotin synthase activity by the Nfs1 protein strongly support the idea that cysteine is the initial sulphur​ donor for biotin in plant mitochondria. The purification of recombinant Adx and AdxR of A. thaliana enables us to establish the first biochemical characterization of a plant Adx/AdxR reaction. These two purified recombinant proteins formed in vitro an efficient low potential electron transfer chain that interacted with the bio2 gene product to reconstitute a functional plant biotin synthase complex. Thus, Bio2 from Arabidopsis is the first identified protein partner in higher plants for this specific mitochondrial redox chain. The electron transfer between the different components of the plant biotin synthase complex requires protein-protein interactions. A preliminary analysis of these interactions was realized. In parallel, we initiated the structural study of the bio2 gene product of A. thaliana in order to complete the functional characterization of biotin synthase reaction in plant cells.
In conclusion, the biochemical characterization of the A. thaliana biotin synthase reaction demonstrates the importance of plant mitochondria in biotin biosynthesis. Moreover, the identification and the involvement of mitochondrial Adx/AdxR redox chain in this vitamin synthesis opens up future prospects in the understanding of the biotin synthase activity regulation in plant cells.

Biotin synthase, Arabidopsis thaliana, Mitochondria, Adrenodoxin, Adrenodoxin reductase, Cysteine desulfurase, Fe-S clusters, Protein-protein interactions