Basic and applied features of multicopper oxidases, CueO, bilirubin oxidase, and laccase.
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 O(2) is converted to H(2)O without releasing activated oxygen species. During the process, O(2) is reduced by MCOs such as lacquer laccase 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 laccase 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.
Division of Material Sciences, Graduate School of Natural Science and Technology, Kanazawa University, Kakuma, Kanazawa 920-1192, Japan. ts0513@kenroku.kanazawa-u.ac.jp
Takeshi
T
Sakurai
Kunishige
K
Kataoka
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D020134:Catalytic Domain; D003300:Copper / Q000737:chemistry; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D019143:Evolution, Molecular; D006801:Humans; D042845:Laccase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D044925:Oxidoreductases Acting on CH-CH Group Donors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D013057:Spectrum Analysis; D013379:Substrate Specificity
D029968:Escherichia coli Proteins; D010940:Plant Proteins; D003300:Copper; D010088:Oxidoreductases; C499679:cueO protein, E coli; D042845:Laccase; D044925:Oxidoreductases Acting on CH-CH Group Donors; C042401:bilirubin oxidase