Upward shift of the pH optimum of Acremonium ascorbate oxidase

Masayasu Sugino , Sachiko Kajita , Koichi Banno , Tsuyoshi Shirai , Takashi Yamane , Masashi Kato , Tetsuo Kobayashi , Norihiro Tsukagoshi
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引用次数: 10

Abstract

A gene encoding a thermostable Acremonium ascorbate oxidase (ASOM) was randomly mutated to generate mutant enzymes with altered pH optima. One of the mutants, which exhibited a significantly higher activity in the pH range 4.5–7 compared to ASOM, had a Gln183Arg substitution in the region corresponding to SBR1, one of the substrate binding regions of the zucchini enzyme. The other mutant with almost the same pH profile as Gln183Arg had a Thr527Ala substitution near the type 3 copper center and became more sensitive to azide than ASOM. Site-directed mutagenesis in the substrate binding regions with reference to the amino acid sequences of plant enzymes led to isolation of mutants shifted upward in the pH optimum; Val193Pro and Val193Pro/Pro190Ile increased the pH optimum by 1 and 0.5 units, respectively, while retaining the near-wild-type thermostability and azide sensitivity. The homology model of ASOM constructed from the zucchini enzyme coordinates suggested that replacement of Val193 by Pro could disturb the ion pair networks among Arg309, Glu192, Arg194 and Glu311. This perturbation could affect either the molecular recognition between the substrate and ASOM or the electron transfer from the substrate to the type 1 copper center, leading to the alkaline shift of the catalytic activity of the mutant enzyme. The other mutations, Val193Pro/Pro190Ile, could also induce similar structural perturbations involving the ion pair networks.

抗坏血酸酯氧化酶最适pH值上移
一个编码耐热性抗坏血酸酯氧化酶(ASOM)的基因随机突变,产生具有改变pH最佳值的突变酶。其中一个突变体在pH值4.5-7范围内的活性明显高于ASOM,在西葫芦酶的底物结合区之一SBR1对应的区域有Gln183Arg取代。另一个pH谱与Gln183Arg几乎相同的突变体在3型铜中心附近有一个Thr527Ala取代,对叠氮化物比ASOM更敏感。参考植物酶的氨基酸序列,在底物结合区进行定点诱变,导致突变体的分离在pH最优位置向上移动;Val193Pro和Val193Pro/Pro190Ile分别提高了最适pH值1和0.5个单位,同时保持了接近野生型的热稳定性和叠氮化物敏感性。利用西葫芦酶座标构建的ASOM同源性模型表明,Pro取代Val193会干扰Arg309、Glu192、Arg194和Glu311之间的离子对网络。这种扰动可能会影响底物与ASOM之间的分子识别或从底物到1型铜中心的电子转移,导致突变酶的催化活性碱性转移。另一个突变,Val193Pro/Pro190Ile,也可以引起类似的涉及离子对网络的结构扰动。
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