Shigeharu Fukuda

Molecular cloning of the second major allergen, Cry j II, from Japanese cedar pollen

Cloning of a cDNA from Cry j II, the second major allergen from Japanese cedar (Cryptomeria japonica) pollen, is described. An isolated Cry j II cDNA contained an open reading frame coding for 514 amino acid residues. The mature Cry j II protein consisted of 388 amino acid residues (R46—S433). According to a homology analysis, no amino acid sequence homology was observed between Cry j II and Cry j I, another major allergen. But Cry j II showed homology with polygalacturonase (PG) derived from tomato (40% identity) at the amino acid level. The sequence information can potentially be used to devise an effective course of immunotherapy for Japanese cedar pollinosis.

Interaction between Trehalose and Alkaline-Earth Metal Ions

We investigated the interaction between trehalose and alkaline-earth metal ions. The nuclear relaxation times of carbon atoms of trehalose were shortened by addition of the alkaline-earth chloride salts, MgCl2, CaCl2, and SrCl2, indicating that trehalose formed metal-complexes with the alkaline-earth metal chlorides. From the data of the 1H–1H coupling constants of trehalose in the presence of the alkaline-earth chlorides, it appeared that trehalose formed complexes with MgCl2, and CaCl2 at the various complexing sites: Mg2+ was coordinated to O-4 and O-4′ of trehalose, and Ca2+ to O-2 and O-3. We succeeded in the preparation of two types of crystals of the trehalose/CaCl2. One was a crystal consisting of trehalose, CaCl2, and water in a ratio of 1:1:1. The other was an anhydrous crystal containing trehalose and CaCl2 in a ratio of 1:2. Several applications of the complexing between trehalose and the metal ions for food processing are proposed.

Structural and mutational analysis of substrate recognition in kojibiose phosphorylase

Glycoside hydrolase (GH) family 65 contains phosphorylases acting on maltose (Glc‐α1,4‐Glc), kojibiose (Glc‐α1,2‐Glc), trehalose (Glc‐α1,α1,‐Glc), and nigerose (Glc‐α1,3‐Glc). These phosphorylases can efficiently catalyze the reverse reactions with high specificities, and thus can be applied to the practical synthesis of α‐glucosyl oligosaccharides. Here, we determined the crystal structures of kojibiose phosphorylase from Caldicellulosiruptor saccharolyticus in complex with glucose and phosphate and in complex with kojibiose and sulfate, providing the first structural insights into the substrate recognition of a glycoside hydrolase family 65 enzyme. The loop 3 region comprising the active site of kojibiose phosphorylase is significantly longer than the active sites of other enzymes, and three residues around this loop, Trp391, Glu392, and Thr417, recognize kojibiose. Various mutants mimicking the residue conservation patterns of other phosphorylases were constructed by mutation at these three residues. Activity measurements of the mutants against four substrates indicated that Trp391 and Glu392, especially the latter, are required for the kojibiose activity.