Monosaccharides and their derivatives which hardly exist in nature are so-called "rare sugars". Rare sugars have significance not only in food industries but also pharmaceutical industries. We discovered a novel L-ribose isomerase from Cellulomonas parahominis (CpL-RbI, 249 amino acids), which catalyzes the reversible isomerization between L-ribose and L-ribulose, L-allose and L-psicose, and D-talose and D-tagatose. Since CpL-RbI has a broad substrate specificity, it is useful for the production of various rare sugars. To elucidate the molecular basis of unique enzymatic properties of CpL-RbI, we determined its crystal structure. The N-terminal His-tagged CpL-RbI overexpressed in Escherichia coli was purified using a nickel affinity column. Crystals of CpL-RbI were obtained from a reservoir solution of 0.1 M sodium acetate trihydrate (pH 4.6) with 3.9 M ammonium acetate, by a hanging-drop vapor-diffusion method at 293 K (Space group C2221, a = 76.8, b = 88.6, c = 152.3 Å). X-ray diffraction data were collected up to 2.10 Å resolution using a Rigaku R-AXIS VII on a RA-Micro7HF rotating anode generator (40 kV, 30 mA) at 100 K. The structure was solved by a molecular replacement method with a structure of Acinetobacter sp L-ribose isomerase (4NS7) as a search model, and refined to R-factor of 0.227. CpL-RbI had a cupin-type beta-barrel structure, and the catalytic site was found between two large beta-sheets with a bound metal ion (Fig. 1). There were two protein molecules in an asymmetric unit, forming a homo-dimer with a non-crystallographic 2-fold symmetry (Fig.1). Furthermore, the PISA server showed that two dimers in crystal were associated to form a stable tetramer. Complex structures with substrates, L-ribose, L-allose and L-psicose, were also successfully determined. We will discuss a broad substrate specificity and catalytic reaction mechanism of CpL-RbI based on its three-dimensional structure.
The Crystal Structure of Mismatch-specific Uracil-DNA Glycosylase (MUG) fromDeinococcus radioduransReveals a Novel Catalytic Residue and Broad Substrate Specificity
