Purification and properties of the chloroplastic form of biotin holocarboxylase synthetase from Arabidopsis thaliana overexpressed in Escherichia coli

Holocarboxylase synthetases (HCSs) are key enzymes in biotin utilisation in both prokaryotes and eukaryotes. In a previous study, we demonstrated that , in plants, HCS activity is localised in cytosol, chloroplasts and mitocho ndria. We also described the cloning and sequencing of a full-length cDNA e ncoding an Arabidopsis thaliana HCS isoform with a putative organelle-trans it peptide. In the study reported here, this cDNA was used to construct an overproducing Escherichia coli strain. The recombinant enzyme was isolated using an efficient three-step purification procedure. Polyclonal antibodies raised against pure HCS were produced to elucidate the subcellular localis ation of this protein. Immunodetection carried out by Western blotting of i solated pea leaf subcellular compartments specifically revealed a single po lypeptide that was ascribed to the chloroplast compartment. Immunocytochemi stry of thin-cut sections from tobacco leaves, transformed by the complete coding sequence of A. thaliana HCS cDNA via Agrobacterium tumefaciens, conf irmed that the enzyme encoded by this cDNA is the chloroplastic isoform. Mo reover, physicochemical, biochemical and kinetic properties of the pure rec ombinant HCS were determined. The native recombinant enzyme is a 37-kDa mon omer. In contrast to the major part of HCS activity measured in leaf extrac ts, the recombinant chloroplastic enzyme did not require addition of Mg2+ t o be fully active, but was substantially inhibited by EDTA. This suggested that the chloroplastic HCS may contain a tightly-bound divalent cation requ ired for enzyme activity. The recombinant enzyme was able to biotinylate ef ficiently apo-biotin carboxyl carrier protein (BCCP) from E. coli and apome thylcrotonoyl-CoA carboxylase (MCCase) from A. thaliana. Apparent K-m value s for the enzyme substrates D-biotin, ATP and apo-MCCase were found to be 1 30 nM, 4.4 mu M and 32 mu M, respectively. Steady-state kinetic analyses of the HCS-catalysed reaction were investigated with respect to reaction mech anism and inhibition by AMP, one of the end-products of the enzyme-catalyse d reaction. Substrate interaction and product inhibition patterns indicated that ATP and D-biotin bind sequentially, in an ordered manner, to the enzy me and that ATP or D-biotin and apo-BCCP bind in ping-pong fashion.