マルチメタルセンターを有する酸化還元酵素の機能研究

取得学位：博士(工学)，学位授与番号：博甲第873号，学位授与年月日：平成19年3月22

Electrochemical characterization of a unique, "neutral" laccase from Flammulina velutipes

The flac1 gene consisted of 1488 bases encodes a novel laccase (Flac1) from Flammulina velutipes. The deduced amino acid sequence of Flac1 with 496 amino acids shows 58-64% homologies with other fungal laccases. The recombinant Flac1 (rFlac1) was heterologously expressed in Pichia pastoris, with sugars of approximately 4 kDa attached on the protein molecule, which has the calculated molecular mass of 53,532 Da. rFlac1 was shown to be a multi-copper oxidase from spectroscopies. The optimum pHs of rFlac1 for oxidations of 2,2\u27-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid), p-phenylenediamine, and o-aminophenol, were 5.0, 5.0, and 6.0-6.5, respectively, showing higher pH values than those from many other fungal laccases. The slightly acidic or neutral optimum pH that is not strongly dependent on substrates is a unique property of rFlac1. Effective O2 reduction was realized by the direct electron transfer of rFlac1 at a highly oriented pyrolytic graphite electrode modified with fine carbon particles (Ketjen Black) in O2-saturated solution. The pHs showing the maximum ΔE°\u27 [= E°\u27(enzyme) - E°\u27(substrate)] coincided well with the optimum pHs shown by rFlac1 under steady-state conditions. The present electrochemical results of rFlac1 indicate that ΔE°\u27 is one of the primary factors to determine the activity of multi-copper oxidases. © 2012 The Society for Biotechnology, Japa

Structure and Function of the Engineered Multicopper Oxidase CueO from Escherichia coli-Deletion of the Methionine-Rich Helical Region Covering the Substrate-Binding Site

金沢大学大学院自然科学研究科物質創成金沢大学理学部CueO is a multicopper oxidase (MCO) that is involved in the homeostasis of Cu in Escherichia coli and is the sole cuprous oxidase to have ever been found. Differing from other MCOs, the substrate-binding site of CueO is deeply buried under a methionine-rich helical region including α-helices 5, 6, and 7 that interfere with the access of organic substrates. We deleted the region Pro357-His406 and replaced it with a Gly-Gly linker. The crystal structures of a truncated mutant in the presence and in the absence of excess Cu(II) indicated that the scaffold of the CueO molecule and metal-binding sites were reserved in comparison with those of CueO. In addition, the high thermostability of the protein molecule and its spectroscopic and magnetic properties due to four Cu centers were also conserved after truncation. As for functions, the cuprous oxidase activity of the mutant was reduced to ca 10% that of recombinant CueO owing to the decrease in the affinity of the labile Cu site for Cu(I) ions, although activities for laccase substrates such as 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid), p-phenylenediamine, and 2,6-dimethoxyphenol increased due to changes in the access of these organic substrates to the type I Cu site. The present engineering of CueO indicates that the methionine-rich α-helices function as a barrier to the access of bulky organic substrates, which provides CueO with specificity as a cuprous oxidase. © 2007 Elsevier Ltd. All rights reserved

Cutoff Values of Serum IgG4 and Histopathological IgG4+ Plasma Cells for Diagnosis of Patients with IgG4-Related Disease

IgG4-related disease is a new disease classification established in Japan in the 21st century. Patients with IgG4-related disease display hyper-IgG4-gammaglobulinemia, massive infiltration of IgG4+ plasma cells into tissue, and good response to glucocorticoids. Since IgG4 overexpression is also observed in other disorders, it is necessary to diagnose IgG4-related disease carefully and correctly. We therefore sought to determine cutoff values for serum IgG4 and IgG4/IgG and for IgG4+/IgG+ plasma cells in tissue diagnostic of IgG4-related disease. Patients and Methods. We retrospectively analyzed serum IgG4 concentrations and IgG4/IgG ratio and IgG4+/IgG+ plasma cell ratio in tissues of 132 patients with IgG4-related disease and 48 patients with other disorders. Result. Serum IgG4 >135  mg/dl demonstrated a sensitivity of 97.0% and a specificity of 79.6% in diagnosing IgG4-related disease, and serum IgG4/IgG ratios >8% had a sensitivity and specificity of 95.5% and 87.5%, respectively. IgG4+cell/IgG+ cell ratio in tissues >40% had a sensitivity and specificity of 94.4% and 85.7%, respectively. However, the number of IgG4+ cells was reduced in severely fibrotic parts of tissues. Conclusion. Although a recent unanimous consensus of all relevant researchers in Japan recently established the diagnostic criteria for IgG4-related disease, findings such as ours indicate that further discussion is needed

The Creation of School Education Bringing up a Student Carrying Tomorrow (3) : The Valuation of "Compulsory Subjects", "Optional Subjects", and "Integrated Subjects"

The purpose of this study is to show the valuation of "Compulsory Subjects", "Optional Subjects", and "Integrated Subjects", to show the relationship between each subjects and "three abilities", "the ability of recognizing othere senses of value", "the ability of self-expression and communication" and "the ability of decision-making" which defined by the project members. The main result of this study is that we should make up the standards which teachers, students and parents recognize as important abilities