※一部利用できない機能があります
| 1.
論文 |
論文
|
Sakurai, Takeshi ; Kataoka, Kunishige
概要:
金沢大学大学院自然科学研究科物質創成<br />The type I copper center in multicopper oxidases is constructed from 1Cys2His and weakly coordin
…
ating 1Met or the non-coordinating 1Phe/1Leu, and it exhibits spectral properties and an alkaline transition similar to those of the blue copper center in blue copper proteins. Since the type I copper center in multicopper oxidases is deeply buried inside the protein molecule, electron transfers to and from type I copper are performed through specific pathways: the hydrogen bond between an amino acid located at the substrate binding site and a His residue coordinating type I copper, and the His-Cys-His sequence connecting the type I copper center and the trinuclear copper center comprised of a type II copper and a pair of type III coppers. The intramolecular electron transfer rates can be tuned by mutating the fourth ligand of type I copper. Further, mutation at the Cys ligand gives a vacant type I copper center and traps the reaction intermediate during the four-electron reduction of dioxygen. © 2007 Birkhäuser Verlag.
続きを見る
|
||||
| 2.
論文 |
論文
|
片岡, 邦重 ; Kataoka, Kunishige ; Tanaka, Kazuhiro ; Sakai, Yoko ; Sakurai, Takeshi
概要:
Bilirubin oxidase (BO) from Myrothecium verrucaria (authentic BO) catalyzing the oxidation of bilirubin to biliverdine w
…
as overexpressed in the methylotrophic yeast, Pichia pastoris. The cDNA encoding BO was cloned into the P. pastoris expression vector pPIC9K under the control of the alcohol oxidase 1 promoter and its protein product was secreted using the Saccharomyces cerevisiae α-mating factor signal sequence. The productivity of recombinant BO (rBO) in P. pastoris was approximately 5000 U/L of culture broth, being about 2.5- and 250-fold higher than rBO expressed in Aspergillus oryzae and S. cerevisiae, respectively. The calculated molecular mass of rBO consisting of 538 amino acids was 60,493 kDa, however, that of SDS–PAGE was 66 kDa because of non-native type N-linked sugar chains. The spectroscopic properties of rBO were typical of multicopper oxidase containing four Cu ions per protein molecule. The specific activity to oxidize bilirubin was 57 U/mg, having a value about twice that of authentic BO and rBO expressed in A. oryzae. Moreover, the thermostability of rBO expressed in P. pastoris was significantly high compared to the authentic BO previously reported. Accordingly, a heterologous expression system of rBO to meet clinical and industrial needs was constructed
続きを見る
|
||||
| 3.
論文 |
論文
|
Sakurai, Takeshi ; Kataoka, Kunishige
概要:
金沢大学大学院自然科学研究科物質創成<br />Multicopper oxidases (MCOs) such as CueO, bilirubin oxidase, and laccase contain four Cu centers
…
, type 1 Cu, type II Cu, and a pair of type III Cu's in a protein molecule consisting of three domains with homologous structure to cupredoxin containing only type I Cu. Type I Cu mediates electron transfer between the substrate and the trinuclear Cu center formed by a type II Cu and a pair of type III Cu's, where the final electron acceptor O2 is converted to H2O without releasing activated oxygen species. During the process, O2 is reduced by MCOs such as lacquer lacease and bilirubin oxidase; the reaction intermediate II with a possible doubly OH--bridged structure in the trinuclear Cu center has been detected. The preceding reaction intermediate I has been detected by the reaction of the lacquer lacease in a mixed valence state, at which type I Cu was cuprous and the trinuclear Cu center was fully reduced, and by the reaction of the Cys → Ser mutant for the type I Cu site in bilirubin oxidase and CueO. An acidic amino acid residue located adjacent to the trinuclear Cu center was proved to function as a proton donor to these reaction intermediates. The substrate specificity of MCO for organic substrates is produced by the integrated effects of the shape of the substrate-binding site and the specific interaction of the substrate with the amino acid located adjacent to the His residue coordinating to the type I Cu. In contrast, the substrate specificity of the cuprous oxidase, CueO, is produced by the segment covering the Cu(I)-binding site so as to obstruct the access of organic substrates. Truncating the segment spanning helix 5 to helix 7 greatly reduced the specificity of CueO for Cu(I) and prominently enhanced the low oxidizing activity for the organic substrates, indicating the success of protein engineering to modify the substrate specificity of MCO. © 2007 The Japan Chemical Journal Forum and Wiley Periodicals, Inc.
続きを見る
|
||||
| 4.
論文 |
論文
|
片岡, 邦重 ; Kataoka, Kunishige ; Tanizawa, Katsuyuki
概要:
The residues L40, A113, V291, and V294, in leucine dehydrogenase (LeuDH), predicted to be involved in recognition of the substrate side chain, have been mutated on the basis of the molecular modeling to mimic the substrate
…
specificities of phenylalanine (PheDH), glutamate (GluDH), and lysine dehydrogenases (LysDH). The A113G and A113G/V291L mutants, imitating the PheDH active site, displayed activities toward -phenylalanine and phenylpyruvate with 1.6 and 7.8% of kcat values of the wild-type enzyme for the preferred substrates, -leucine and its keto-analog, respectively. Indeed, the residue A113, corresponding to G114 in PheDH, affects the volume of the side-chain binding pocket and has a critical role in discrimination of the bulkiness of the side chain. Another two sets of mutants, substituting L40 and V294 of LeuDH with the corresponding residues predicted in GluDH and LysDH, were also constructed and characterized. Emergence of GluDH and LysDH activities in L40K/V294S and L40D/V294S mutants, respectively, indicates that the two corresponding residues in the active site of amino acid dehydrogenases are important for discrimination of the hydrophobicity/polarity of the aliphatic substrate side chain. All these results demonstrate that the substrate specificities of the amino acid dehydrogenases can be altered by protein engineering. The engineered dehydrogenases are expected to be used for production and detection of natural and non-natural amino acids.
続きを見る
|
||||
| 5.
論文 |
論文
|
片岡, 邦重 ; Kataoka, Kunishige ; Nakai, Masami ; Yamaguchi, Kazuya ; Suzuki, Shinnichiro
概要:
The gene coding for the 109-amino-acid, non-glycosylated form of mavicyanin was synthesized and expressed inEscherichia
…
coli.The recombinant protein refolded fromE. coliinclusion bodies was purified and characterized. Its spectroscopic properties are fully identical to those of mavicyanin isolated from zucchini, even in the absence of its carbohydrate moiety. The blue cooper center of mavicyanin strongly binds three ligands (2His and Cys) as well as many blue copper proteins. To disclose the fourth ligand of mavicyanin, Met was substituted for Gln95 by site-directed mutagenesis. The replacement changes from a rhombic EPR signal to an axial one and exhibits the quite similar absorption and CD spectra to those of plastocyanin. The midpoint potential of Gln95→Met mavicyanin shows the positive shift of 187 mV compared with the recombinant protein, being close to the values of plastocyanins. The differences of the spectroscopic and electrochemical properties between mavicyanin and its mutant demonstrate that the fourth ligand of mavicyanin is Gln95.
続きを見る
|
||||
| 6.
論文 |
論文
|
Kataoka, Kunishige ; Komori, Hirofumi ; Ueki, Yusaku ; Konno, Yusuke ; Kamitaka, Yuji ; Kurose, Shinji ; Tsujimura, Seiya ; Higuchi, Yoshiki ; Kano, Kenji ; Seo, Daisuke ; Sakurai, Takeshi
概要:
金沢大学大学院自然科学研究科物質創成<br />金沢大学理学部<br />CueO is a multicopper oxidase (MCO) that is involved in the homeostasis of Cu in Es
…
cherichia 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.
続きを見る
|
||||
| 7.
論文 |
論文
|
Seo, Daisuke ; Okabe, Seisuke ; Yanase, Mitsuhiro ; Kataoka, Kunishige ; Sakurai, Takeshi
概要:
金沢大学理工研究域物質化学系<br />Ferredoxin-NADP+ oxidoreductases (FNRs) of Bacillus subtilis (YumC) and Rhodopseudomonas palustris C
…
GA009 (RPA3954) belong to a novel homo-dimeric type of FNR with high amino acid sequence homology to NADPH-thioredoxin reductases. These FNRs were purified from expression constructs in Escherichia coli cells, and their steady-state reactions with [2Fe-2S] type ferredoxins (Fds) from spinach and R. palustris, [4Fe-4S] type Fd from B. subtilis, NAD(P)+/NAD(P)H and ferricyanide were studied. From the Km and kcat values for the diaphorase activity with ferricyanide, it is demonstrated that both FNRs are far more specific for NADPH than for NADH. The UV-visible spectral changes induced by NADP+ and B. subtilis Fd indicated that both FNRs form a ternary complex with NADP+ and Fd, and that each of the two ligands decreases the affinities of the others. The steady-state kinetics of NADPH-cytochrome c reduction activity of YumC is consistent with formation of a ternary complex of NADPH and Fd during catalysis. These results indicate that despite their low sequence homology to other FNRs, these enzymes possess high FNR activity but with measurable differences in affinity for different types of Fds as compared to other more conventional FNRs. © 2008 Elsevier B.V. All rights reserved.
続きを見る
|
||||
| 8.
論文 |
論文
|
Kajikawa, Takao ; Kataoka, Kunishige ; Sakurai, Takeshi
概要:
CueO has a branched hydrogen bond network leading from the exterior of the protein molecule to the trinuclear copper cen
…
ter. This network transports protons in the four-electron reduction of dioxygen. We replaced the acidic Glu506 and Asp507 residues with the charged and uncharged amino acid residues. Peculiar changes in the enzyme activity of the mutants relative to the native enzyme indicate that an acidic amino acid residue at position 506 is essential for effective proton transport. The Ala mutation resulted in the formation of a compensatory hydrogen bond network with one or two extra water molecules. On the other hand, the Ile mutation resulted in the complete shutdown of the hydrogen bond network leading to loss of enzymatic activities of CueO. In contrast, the hydrogen bond network without the proton transport function was constructed by the Gln mutation. These results exerted on the hydrogen bond network in CueO are discussed in comparison with proton transfers in cytochrome oxidase. © 2012 Elsevier Inc.
続きを見る
|
||||
| 9.
論文 |
論文
|
Saito-Otsuka, Kaori ; Kurose, Shinji ; Tsujino, Yoshio ; Osakai, Toshiyuki ; Kataoka, Kunishige ; Sakurai, Takeshi ; Tamiya, Eiichi
概要:
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'-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°' [= E°'(enzyme) - E°'(substrate)] coincided well with the optimum pHs shown by rFlac1 under steady-state conditions. The present electrochemical results of rFlac1 indicate that ΔE°' is one of the primary factors to determine the activity of multi-copper oxidases. © 2012 The Society for Biotechnology, Japan
続きを見る
|
||||
| 10.
論文 |
論文
|
Kajikawa, Takao ; Kataoka, Kunishige ; Sakurai, Takeshi
概要:
CueO has a branched hydrogen bond network leading from the exterior of the protein molecule to the trinuclear copper cen
…
ter. This network transports protons in the four-electron reduction of dioxygen. We replaced the acidic Glu506 and Asp507 residues with the charged and uncharged amino acid residues. Peculiar changes in the enzyme activity of the mutants relative to the native enzyme indicate that an acidic amino acid residue at position 506 is essential for effective proton transport. The Ala mutation resulted in the formation of a compensatory hydrogen bond network with one or two extra water molecules. On the other hand, the Ile mutation resulted in the complete shutdown of the hydrogen bond network leading to loss of enzymatic activities of CueO. In contrast, the hydrogen bond network without the proton transport function was constructed by the Gln mutation. These results exerted on the hydrogen bond network in CueO are discussed in comparison with proton transfers in cytochrome oxidase. © 2012 Elsevier Inc.
続きを見る
|
