Myrothecium verrucaria bilirubin oxidase and its mutants for potential copper ligands
Myrothecium verrucaria bilirubin oxidase and its mutants for potential copper ligands
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DOI：
10.1021/bi9819531
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发表时间：
1999-03-09
期刊：
影响因子：
2.9
通讯作者：
Samejima, T
中科院分区：
文献类型：
--
作者：
Shimizu, A;Kwon, JH;Samejima, T
从疣状漆斑菌 MT-1（正宗酶）培养基中纯化的胆红素氧化酶（EC：1.3.3.5）在体外催化胆红素氧化为胆绿素，并通过使用带有表达载体的米曲霉的胆红素氧化酶基因过表达系统获得重组酶（野生型）。吸收光谱和ESR光谱显示，两种胆红素氧化酶都是含有1型、2型和3型铜的多铜氧化酶，类似于漆酶、抗坏血酸氧化酶和铜蓝蛋白。已对每种类型铜的可能配体进行了定点诱变。在一些突变体中，Cys457 --> Val、Ala、His94 --> Val 和 His134.136 --> Val，1 型和 2 型铜中心完全受到干扰，酶活性完全丧失。与全酶不同，这些突变体显示出3型铜信号。然而，即使通过突变 1 型铜配体之一（即突变体 Met467 --> Gly），1 型铜的光学和磁特性特征仍然保留，表现出微弱但明显的酶活性。双突变体 His456.458 --> Val 仅具有 1 型 Cu，在 600 nm 处显示蓝色条带（epsilon = 1.6 x 10(3)），并且 ESR 信号具有非常窄的超精细分裂（A（平行于）= 7.2 x 10(-3) cm(-1)）。由于不存在 2 型和 3 型铜，因此突变体不显示酶活性。这些结果强烈暗示胆红素氧化酶中的特殊序列His456-Cys457-His458在1型铜位点和由2型和3型铜位点组成的三核中心之间形成分子内电子转移途径。
Bilirubin oxidase (EC:1.3.3.5) purified from a culture medium of Myrothecium verrucaria MT-1 (authentic enzyme) catalyzes the oxidation of bilirubin to biliverdin in vitro and recombinant enzyme (wild type) was obtained by using an overexpression system of the bilirubin oxidase gene with Aspergillus oryzae harboring an expression vector. The absorption and ESR spectra showed that both bilirubin oxidases are multicopper oxidases containing type 1, type 2, and type 3 coppers similar to laccase, ascorbate oxidase, and ceruloplasmin. Site-directed mutagenesis has been performed for the possible ligands of each type of copper. In some mutants, Cys457 --> Val, Ala, His94 --> Val, and His134.136 --> Val, type 1 and type 2 copper centers were perturbed completely and the enzyme activity was completely lost. Differing from the holoenzyme, these mutants showed type 3 copper signals. However, the optical and magnetic properties characteristic of type 1 copper were retained even by mutating one of the type 1 copper ligands, i.e., a mutant, Met467 --> Gly, showed a weak but apparent enzyme activity. A double mutant His456.458 --> Val had only type 1 Cu, showing a blue band at 600 nm (epsilon = 1.6 x 10(3)) and an ESR signal with very narrow hyperfine splitting (A(parallel to) = 7.2 x 10(-3) cm(-1)). Since the type 2 and type 3 coppers are not present, the mutant did not show enzyme activity. These results strongly imply that the peculiar sequence in bilirubin oxidase, His456-Cys457-His458, forms an intramolecular electron-transfer pathway between the type 1 copper site and the trinuclear center composed of the type 2 and type 3 copper sites.