Department of Bioscience, Tokyo University of Agriculture* Division of Integrative Life Sciences, Graduate School of Life and Environmental Sciences, University of Tsukuba**
○Daisuke Iino* Yasuaki Takakura** Mika Kuroiwa* Yasuyuki Sasaki* Takayuki Hoshino** Kanju Ohsawa* Akira Nakamura** Shunsuke Yajima*
Bacteria elaborate the systems to survive antibiotics in various ways, and in turn, by using them the selection markers to clone genes have been developed as tools for molecular biology. Hygromycin B is one of the aminoglycoside antibiotics and targets ribosome to kill eukaryotic cells as well as prokaryotic ones. On the other hand, hygromycin B phosphotransferase (Hph) inactivates this antibiotic using ATP, thus this gene, especially that from Escherichia coli, has been used as a selection marker in many host-vector systems, including plants and animals. However, the tertiary structure of this protein has never been reported.
Recently, we have obtained a mutant Hph useful as a selection marker in a thermophilic bacterium, Thermus thermophilus, by the directed evolution1). The mutant protein, Hph5, contains five amino acid substitutions, and shows an increased thermostability of about 16 deg in vivo and in vitro, compared with the wild type protein.
The Se-Met substituted protein was crystallized using the hanging-drop vapour-diffusion method. The crystal grew to dimensions of 0.2 x 0.2 x 0.2 mm in a couple of days. The data was collected at 2.1-Å resolution at BL-5A, Photon Factory (Tsukuba, Japan). The crystal belongs to space group P3221, with unit cell parameters of a = b = 71.0 Å, c = 125.0 Å, and an asymmetric unit contains one monomer of Hph5 (340 residues). The data was processed by Mosflm, and the initial phase was obtained using the program SOLVE followed by the density modification with RESOLVE. RESOLVE also successfully traced the density to build the initial model with about 70% of total residues. Further modeling and refinement are currently continuing.
1) Nakamura, A et al. J Biosci Bioeng 2005, 100,158-63.