Basic and applied features of multicopper oxidases, cueo, bilirubin oxidase, and laccase

金沢大学大学院自然科学研究科物質創成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\u27s 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\u27s, 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

Structure and function of type I copper in multicopper oxidases

金沢大学大学院自然科学研究科物質創成The type I copper center in multicopper oxidases is constructed from 1Cys2His and weakly coordinating 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

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

金沢大学理工研究域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。研究課題/領域番号:14780487, 研究期間(年度):2002-2003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

Modifications on the hydrogen bond network by mutations of Escherichia coli copper efflux oxidase affect the process of proton transfer to dioxygen leading to alterations of enzymatic activities

CueO has a branched hydrogen bond network leading from the exterior of the protein molecule to the trinuclear copper center. 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

A novel resting form of the trinuclear copper center in the double mutant of a multicopper oxidase, CueO, Cys500Ser/Glu506Ala

A multicopper oxidase, CueO was doubly mutated at its type I copper ligand, Cys500 and an acidic amino acid residue located 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.Embargo Period 24 month

Alteration of substrate specificity of leucine dehydrogenase by site-directed mutagenesis

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

Study on dioxygen reduction by mutational modifications of the hydrogen bond network leading from bulk water to the trinuclear copper center in bilirubin oxidase

The hydrogen bond network leading from bulk water to the trinuclear copper center in bilirubin oxidase is constructed with 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

An O-centered structure of the trinuclear copper center in the Cys500Ser/Glu506Gln mutant of CueO and structural changes in low to high X-ray dose conditions

Right on CueO: The O-centered structure of the trinuclear copper center in a multicopper oxidase (CueO) was shown to be 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

Studies of interaction of homo-dimeric ferredoxin-NAD(P)+ oxidoreductases of Bacillus subtilis and Rhodopseudomonas palustris, that are closely related to thioredoxin reductases in amino acid sequence, with ferredoxins and pyridine nucleotide coenzymes

金沢大学理工研究域物質化学系Ferredoxin-NADP+ oxidoreductases (FNRs) of Bacillus subtilis (YumC) and Rhodopseudomonas palustris CGA009 (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