The effects of the site-directed removal of N-glycosylation from cationic peanut peroxidase on its function
The effects of the site-directed removal of N-glycosylation from cationic peanut peroxidase on its function
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DOI：
10.1006/abbi.2000.2187
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发表时间：
2001-02-01
影响因子：
3.9
通讯作者：
van Huystee, RB
中科院分区：
文献类型：
--
作者：
Bao, LG;Ma, SW;van Huystee, RB
花生过氧化物酶已被衍射，其血红素和钙部分的位置已被显示，并证明其作用。然而，其聚糖的结构和作用现在才被阐明。研究了3种N-连接复合聚糖对花生（Arachis hypogaea L cv.）瓦伦西亚），如在转基因烟草中由prxPNC 1表达的，通过分别用Q定点置换三个糖基化位点N-60、N-144和N-185中的每一个来分析。在转基因烟草中表达了带有3'组氨酸标签的突变体prxPNC 1 cDNA。从转基因烟草细胞悬浮培养物的培养基中分离的突变过氧化物酶的催化能力，热稳定性，和去折叠特性的效果进行了比较与野生型cPrx从花生。发现N-60和N-144处聚糖的消融影响cPrx催化能力的完全表达。N-185处的聚糖对热稳定性很重要，N-185处的碳水化合物链的去除也很重要，导致在50 ℃的温度下酶活性迅速降低。所有三种聚糖似乎都影响蛋白质的折叠。(C)北京：科学出版社.
Peanut peroxidase has been diffracted, The location of its heme and calcium moieties have been shown and their role demonstrated. However, the structure and role of its glycans is only now being elucidated. The role of three N-linked complex glycans on cationic peroxidase (cPrx) of peanut (Arachis hypogaea L cv. Valencia), as expressed by prxPNC1 in transgenic tobacco, was analyzed by site-directed replacement of each of the three glycosylation sites, N-60, N-144, and N-185 with Q, individually. The mutant prxPNC1 cDNAs with a 3' histidine-tag were expressed in transgenic tobacco. The effect on the catalytic ability, thermal stability, and unfolding properties of the mutant peroxidases, isolated from the medium of transgenic tobacco cell suspension cultures were compared with those of the wild cPrx from peanut. It was found that the ablation of the glycans at N-60 and N-144 influences the full expression of the cPrx catalytic ability. The glycan at N-185 is important for the thermostability, as is the removal of the carbohydrate chain at N-185, resulting in rapid enzymatic decrease at temperatures of 50 degreesC. All three glycans appeared to influence the folding of the protein. (C) 2001 Academic Press.