Site-directed mutagenesis study of yeast peptide:: N-glycanase -: Insight into the reaction mechanism of deglycosylation
Site-directed mutagenesis study of yeast peptide:: N-glycanase -: Insight into the reaction mechanism of deglycosylation
复制标题
DOI：
10.1074/jbc.m111383200
复制
发表时间：
2002-04-12
影响因子：
4.8
通讯作者：
Lennarz, WJ
中科院分区：
文献类型：
--
作者：
Katiyar, S;Suzuki, T;Lennarz, WJ
酵母肽：N-聚糖酶（Png 1 p; PNGase），一种参与蛋白质的蛋白酶体依赖性降解的去糖基化酶，已被报道为基于序列比对的转氨酶超家族的成员。在本研究中，我们研究了Png 1 p的结构与功能的关系，通过定点突变。Png 1 p的Cys-191、His-218和Asp-235在因子XIIIa的序列中是保守的，其中这些氨基酸构成催化三联体。Png 1 p中这些残基的点突变导致活性完全丧失，这与每个残基在催化糖蛋白去糖基化中的作用一致。其他保守的氨基酸残基，Trp-220，Trp-231，Arg-210，和Glu-222，也是至关重要的折叠和结构的稳定性的酶所揭示的圆二色性分析。通过绘制已知因子XIIIa三维结构内Png 1 p的保守氨基酸来预测突变的潜在影响。我们的数据表明，缺乏酶活性时，任何催化三联体残基突变是由于在三联体的情况下，或由于破坏的天然折叠的酶的电荷中继的情况下。这些发现有力地表明PNGases和转氨酶具有共同的进化谱系。
Yeast peptide:N-glycanase (Png1p; PNGase), a deglycosylation enzyme involved in the proteasome dependent degradation of proteins, has been reported to be a member of the transglutaminase superfamily based on sequence alignment. In this study we have investigated the structure-function relationship of Png1p by site-directed mutagenesis. Cys-191, His-218, and Asp-235 of Png1p are conserved in the sequence of factor XIIIa, where these amino acids constitute a catalytic triad. Point mutations of these residues in Png1p resulted in complete loss in activity, consistent with a role for each in catalyzing deglycosylation of glycoproteins. Other conserved amino acid residues, Trp-220, Trp-231, Arg-210, and Glu-222, were also vitally important for folding and structure stability of the enzyme as revealed by circular dichroism analysis. The potential effects of the mutations were predicted by mapping the conserved amino acids of Png1p within the known three-dimensional structure of factor XIIIa. Our data suggest that the lack in enzyme activity when any of the catalytic triad residues is mutated is either due to the absence of charge relay in the case of the triad or due to the disruption of the native fold of the enzyme. These findings strongly suggest a common evolutionary lineage for the PNGases and transglutaminases.