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| 1.
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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
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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.
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| 2.
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片岡, 邦重 ; Kataoka, Kunishige ; Tanaka, Kazuhiro ; Sakai, Yoko ; Sakurai, Takeshi
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Bilirubin oxidase (BO) from Myrothecium verrucaria (authentic BO) catalyzing the oxidation of bilirubin to biliverdine w
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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
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| 3.
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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
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, 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.
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| 4.
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片岡, 邦重 ; Kataoka, Kunishige ; Tanizawa, Katsuyuki
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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
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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.
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| 5.
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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
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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.
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| 6.
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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
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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.
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| 7.
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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
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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.
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| 8.
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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
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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.
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| 9.
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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
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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
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| 10.
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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
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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.
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| 11.
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Komori, Hirofumi ; Sugiyama, Ryosuke ; Kataoka, Kunishige ; Higuchi, Yoshiki ; Sakurai, Takeshi
概要:
Right on CueO: The O-centered structure of the trinuclear copper center in a multicopper oxidase (CueO) was shown to be
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an intermediate of the four-electron reduction of dioxygen (see picture). This structure was determined by in situ data collection of X-ray diffractions and copper K-edge spectra at low to high X-ray dose conditions. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
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| 12.
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Komori, Hirofumi ; Sugiyama, Ryosuke ; Kataoka, Kunishige ; Miyazaki, Kentaro ; Higuchi, Yoshiki ; Sakurai, Takeshi
概要:
Structural models determined by X-ray crystallography play a central role in understanding the catalytic mechanism of en
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zymes. However, X-ray radiation generates hydrated electrons that can cause significant damage to the active sites of metalloenzymes. In the present study, crystal structures of the multicopper oxidases (MCOs) CueO from Escherichia coli and laccase from a metagenome were determined. Diffraction data were obtained from a single crystal under low to high X-ray dose conditions. At low levels of X-ray exposure, unambiguous electron density for an O atom was observed inside the trinuclear copper centre (TNC) in both MCOs. The gradual reduction of copper by hydrated electrons monitored by measurement of the Cu K-edge X-ray absorption spectra led to the disappearance of the electron density for the O atom. In addition, the size of the copper triangle was enlarged by a two-step shift in the location of the type III coppers owing to reduction. Further, binding of O2 to the TNC after its full reduction was observed in the case of the laccase. Based on these novel structural findings, the diverse resting structures of the MCOs and their four-electron O2-reduction process are discussed. © 2014 International Union of Crystallography.
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| 13.
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Kurose, Shinji ; Kataoka, Kunishige ; Shinohara, Naoya ; Miura, Yuko ; Tsutsumi, Maiko ; Tsujimura, Seiya ; Kano, Kenji ; Sakurai, Takeshi
概要:
Replacement of Met510, the axial ligand to the type I Cu in a cuprous oxidase CueO, with Leu afforded the three-coordinated type I Cu, while Gln, Ala, and Thr mutations led to the replacement of the thioether ligand with oxygen ligands
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(amide carbonyl group and water), and characteristic properties of absorption, circular dichroism, and electron paramagnetic resonance spectra of a variety of Met510 mutants were correlated with the changes in redox potential and enzyme activities.
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| 14.
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Morishita, Hirotoshi ; Kurita, Daisuke ; Kataoka, Kunishige ; Sakurai, Takeshi
概要:
The hydrogen bond network leading from bulk water to the trinuclear copper center in bilirubin oxidase is constructed wi
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th Glu463 and water molecules to transport protons for the four-electron reduction of dioxygen. Substitutions of Glu463 with Gln or Ala were attributed to virtually complete loss or significant reduction in enzymatic activities due to an inhibition of the proton transfer steps to dioxygen. The single turnover reaction of the Glu463Gln mutant afforded the highly magnetically interacted intermediate II (native intermediate) with a broad g = 1.96 electron paramagnetic resonance signal detectable at cryogenic temperatures. Reactions of the double mutants, Cys457Ser/Glu463Gln and Cys457Ser/Glu463Ala afforded the intermediate I (peroxide intermediate) because the type I copper center to donate the fourth electron to dioxygen was vacant in addition to the interference of proton transport due to the mutation at Glu463. The intermediate I gave no electron paramagnetic resonance signal, but the type II copper signal became detectable with the decay of the intermediate I. Structural and functional similarities between multicopper oxidases are discussed based on the present mutation at Glu463 in bilirubin oxidase. © 2014 Elsevier Inc. All rights reserved.
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| 15.
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Kajikawa, Takao ; Sugiyama, Ryosuke ; Kataoka, Kunishige ; Sakurai, Takeshi
概要:
A multicopper oxidase, CueO was doubly mutated at its type I copper ligand, Cys500 and an acidic amino acid residue loca
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ted in the proton transfer pathway, Glu506, to Ser and Ala, respectively. Cys500Ser/Glu506Ala was mainly in a novel resting form to afford the absorption band at ca. 400. nm and an EPR signal with a highly anisotropic character derived from type III copper. However, Cys500Ser/Glu506Ala gave the same reaction intermediate (peroxide intermediate) as that from Cys500Ser and Cys500Ser/Glu506Gln. © 2015 Elsevier Inc.<br />Embargo Period 24 months
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| 16.
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Kataoka, Kunishige ; Sugiyama, Ryosuke ; Hirota, Shun ; Inoue, Megumi ; Urata, Kanae ; Minagawa, Yoichi ; Seo, Daisuke ; Sakurai, Takeshi
概要:
金沢大学理工研究域物質化学系<br />The mechanism of the four-electron reduction of dioxygen by a multicopper oxidase, CueO, was studied
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based on reactions of single and double mutants with Cys500, a type I copper ligand, and the noncoordinating Asp112 and Glu506, which form hydrogen bonds with the trinuclear copper center directly and indirectly via a water molecule. The reaction of C500S containing a vacant type I copper center produced intermediate I in an EPR-silent peroxide-bound form. The formation of intermediate I from C500S/D112N was restricted due to a reduction in the affinity of the trinuclear copper center for dioxygen. The state of intermediate I was realized to be the resting form of C500S/E506Q and C500S of the truncated mutant Δα5-7CueO, in which the 50 amino acids covering the substrate-binding site were removed. Reactions of the recombinant CueO and E506Q afforded intermediate II, a fully oxidized form different from the resting one, with a very broad EPR signal, g < 2, detectable only at cryogenic temperatures and unsaturated with high power microwaves. The lifetime of intermediate II was prolonged by the mutation at Glu506 involved in the donation of protons. The structure of intermediates I and II and the mechanism of the four-electron reduction of dioxygen driven by Asp112 and Glu506 are discussed. © 2009 by The American Society for Biochemistry and Molecular Biology, Inc.
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| 17.
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片岡, 邦重 ; Kataoka, Kunishige
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金沢大学理工研究域物質化学系<br />二臭化インディゴの分子構造をもつ貝紫は，紀元前1,600 年以前から使用されている希少な動物性天然染料である。本研究では，アカニシガイの鰓下腺から貝紫色素前駆体の臭素化反応を触媒するブロモペルオキシダ
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ーゼ(BPO)の精製を試みた。BPOは鰓下腺に微量しか存在していないため，均一精製には至らなかったが，膜画分に存在するBPOの活性発現に，可溶性活性化タンパク質が必要であることを明らかにした。２成分で構成されるBPOはこれまでに報告例のない新規の酵素である。<br />Tyrian purple having a molecular structure of 6,6'-dibromoindigo is a rare animal natural dye used since 1,600 BC. In this study, we tried to purify bromoperoxidase (BPO), which catalyzes the bromination reaction of purple pigment precursor from the hypobranchial gland of the shellfish, Rapana venosa. Since BPO exists only in trace amounts in shellfish, it could not be purified homogeneously, but revealed that a soluble activating protein is necessary for the activity of BPO present in the membrane fraction. BPO composed of two components is a novel enzyme, which has never been seen before.<br />研究課題/領域番号:15K14709, 研究期間(年度):2015-04-01 - 2018-03-31
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| 18.
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片岡, 邦重 ; Kataoka, Kunishige
概要:
バイオ燃料電池の実用化に必須である白金代替酸素還元触媒の創成を目的として，酸素の４電子還元反応を触媒するマルチ銅オキシダーゼをターゲットに，高反応性酵素の探索と既存酵素のタンパク質工学的高機能化を行った。探索研究では，嫌気性細菌のラッカーゼ
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と，昆虫のクチクラ硬化ラッカーゼ遺伝子の発現系を構築し，前者が既存酵素と全く異なる性質を持つ事を明らかにした。さらに，微生物由来の既存酵素をベースとしたタンパク質工学的改変により，酸素還元活性，電極反応性の向上に成功した。<br />For the development of the platinum-substitute anodic enzyme catalysts for bio-fuel cell, I performed the in silico screening to find new multi-copper oxidases and the mutations against the common enzymes to improve their oxygen reduction activity. In the screening for the new multi-copper oxidases, I found two laccase genes from an anaerobic bacterium and an insect. The heterologous expression systems for the laccase genes were successfully constructed, and the recombinant laccase from anaerobe was found to have unreported properties so far. Furthermore, the improvements of the bacterial common enzymes in the oxygen-reduction activity and the electrode response were achieved by protein engineering.<br />研究課題/領域番号:23580131, 研究期間(年度):2011-2013
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| 19.
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片岡, 邦重 ; Kataoka, Kunishige
概要:
プロトンポンプの分子進化過程を明らかにするために,一酸化窒素還元酵素(NOR)の異種発現系を構築し,ヘムb3-FeB複核活性中心を構成するFeB配位残基に変異を導入した。Glu190, Glu194及びGlu259を対象にAla, Asp,
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Gln置換変異体を作製したところ,何れの変異体も複核中心の形成が阻害され,これらの残基がNORの活性中心形成に重要であることが明らかになった。また,NORの生理的電子供与体を検索し,3種類の可溶性Cytcを単離することに成功した。<br />研究課題/領域番号:19570125, 研究期間(年度):2007-2009
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| 20.
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片岡, 邦重 ; Kataoka, Kunishige
概要:
マルチ銅オキシダーゼ（MCO）は，基質を酸化した電子を用いて酸素を４電子還元し水を生成する反応を触媒する。本研究では，MCOの一種である大腸菌の一価銅酸化酵素CueOを対象に，酸素還元反応中間体の構造解析を目指して研究を実施した。三重変異の
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導入により中間体を安定化させることに成功し，中性子線回折に使用できる良質で大きな結晶を再現性良く作製した。また，進化分子工学的手法を用いて6種類の新規高活性型アミノ酸置換体を取得した。一部は構造解析にも成功し，ドメイン3のタワーループ領域の不安定化が活性増大を招くことを明らかにした。本研究は，MCOの反応制御に新たな方法論を与え，その産業応用に貢献する。<br />Multicopper oxidases (MCOs) catalyze the oxidation of a wide range of substrates by reducing O2 to H2O without releasing activated oxygen species. In this study, we aimed to elucidate the structure of the 4-electron reduction intermediate of oxygen using the cuprous oxidase CueO from Escherichia coli. We succeeded in stabilizing the intermediate by introducing a triple mutation, and produced a high-quality crystal that can be used for neutron diffraction. In addition, directed evolution has yielded six very active mutants. Some also succeeded in structural analysis, and revealed that destabilization of the tower loop region of domain 3 leads to increased activity. These results provide a new methodology for controlling the activity of MCOs and contribute to their industrial application.<br />研究課題/領域番号:18K05432, 研究期間(年度):2018-04-01 - 2021-03-31<br />出典：「反応中間体の構造解析によるマルチ銅オキシダーゼの酸素還元機構の解明と応用」研究成果報告書 課題番号18K05432（KAKEN：科学研究費助成事業データベース（国立情報学研究所）） （https://kaken.nii.ac.jp/report/KAKENHI-PROJECT-18K05432/18K05432seika/）を加工して作成
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| 21.
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片岡, 邦重 ; Kataoka, Kunishige
概要:
金沢大学理工研究域<br />Halomonas halodenitrificans(旧称Palacoccus halodenitrificans)由来の一酸化窒素還元酵素(NOR)の活性中心を含むNorBサブユニットをコードするnorB遺
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伝子をpTrc99Aベクターのtrcプロモーター制御下に連結した発現プラスミドを作製し、ヘム合成能が増強された大腸菌Escharichia coli JCB7120株をホストに用いてNorBサブユニットの異種発現を行った。嫌気培養によりNorBを発現させた菌体から超音波破砕と超遠心分離により膜画分を調整し、Nonyl-β-D-glucosideを用いて膜タンパク質を可溶化後、陰イオン交換クロマトグラフィーにより組換え型NorBを部分精製し、その分光学的性質を野生型NORと比較した。NorB部分精製標品の吸収スペクトルでは還元型で558,527,422nmにそれぞれα,β,γ吸収帯を示し、MCDスペクトルでは酸化体のソーレ領域(420nm)と還元型のQバンド領域(560nm)に低スピンヘム鉄の吸収と、還元型のソーレ領域(430nm)に高スピンヘム鉄に特徴的な吸収が観察された。これらの吸収は野生型NORの低スピンヘムbおよび高スピンヘムb_3の吸収とよく類似していた。従って大腸菌で異種発現した組換え型NorBは野生型NORと同様に2種のB型ヘムを含むことが明らかになった。また、組換え型NorBのESRスペクトルではg=3.14,2.27に低スピンヘム鉄(III)に特徴的な斜方対称のシグナルと、g=5.99に高スピンヘム鉄(III)のシグナルが確認できたが、非ヘム鉄の存在を示唆するg=4.26のシグナル強度が野生型NORの対応するシグナルより強く、ヘムb_3-非ヘム鉄からなる複核中心の酸素架橋構造に違いがあると考えられる。<br />研究課題/領域番号:14780487, 研究期間(年度):2002-2003<br />出典：「一酸化窒素還元酵素の活性中心構造と触媒反応機構の解明」研究成果報告書 課題番号14780487（KAKEN：科学研究費助成事業データベース（国立情報学研究所））（https://kaken.nii.ac.jp/ja/grant/KAKENHI-PROJECT-14780487/)を加工して作成
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