Involvement of a conserved tryptophan residue in the UDP-glucose binding of large clostridial cytotoxin glycosyltransferases
Involvement of a conserved tryptophan residue in the UDP-glucose binding of large clostridial cytotoxin glycosyltransferases
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DOI：
10.1074/jbc.275.18.13228
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发表时间：
2000-05-05
影响因子：
4.8
通讯作者：
Aktories, K
中科院分区：
文献类型：
--
作者：
Busch, C;Hofmann, F;Aktories, K
大的梭状芽胞杆菌细胞毒素以UDP-葡萄糖为共底物催化Rho/Ras GTP酶的糖基化。通过对梭状芽胞杆菌致死毒素和艰难梭菌毒素B片段的定点突变，我们确定位于所有梭状芽胞杆菌细胞毒素催化区域的保守区域的色氨酸102对UDP-葡萄糖结合是至关重要的。在微量注射后，Trp-102与丙氨酸交换使葡萄糖转移酶活性降低约1000倍并阻断细胞毒活性，酪氨酸取代Trp-102导致酶活性降低100倍，表明酪氨酸部分补偿色氨酸功能，葡萄糖转移酶和糖水解酶活性降低主要是由于这些突变体对UDP-葡萄糖的K-m增加所致。这些数据表明，大的梭状芽胞杆菌细胞毒素与共底物UDP-葡萄糖的结合与保守的色氨酸残基有关。数据库搜索显示，不同的蛋白质组共享最近确定的DXD基序(Busch，C.，Hofmann，F.，Selzer，J.，Munro，J.，Jeckel，D.，和Akurents，K.(1998)J.Biol)。化学。19566-19572)和一个由色氨酸残基定义的保守区域，该区域相当于索氏杆菌致死毒素的Trp-102。根据我们的发现，我们提出了一个新的糖基转移酶家族，包括原核和真核蛋白。
Large clostridial cytotoxins catalyze the glucosylation of Rho/Ras GTPases using UDP-glucose as a cosubstrate. By site-directed mutagenesis of Clostridium sordellii lethal toxin and Clostridium difficile toxin B fragments, we identified tryptophan 102, which is located in a conserved region within the catalytic domain of all clostridial cytotoxins, to be crucial for UDP-glucose binding. Exchange of Trp-102 with alanine decreased the glucosyltransferase activity by about 1,000-fold and blocked cytotoxic activity after microinjection, Replacement of Trp-102 by tyrosine caused a 100-fold reduction in enzyme activity, indicating a partial compensation of the tryptophan function by tyrosine, Decrease in glucosyltransferase and glycohydrolase activity was caused predominantly by an increase in the K-m for UDP-glucose of these mutants. The data indicate that the conserved tryptophan residue is implicated in the binding of the cosubstrate UDP-glucose by large clostridial cytotoxins. Data bank searches revealed different groups of proteins sharing the recently identified DXD motif (Busch, C., Hofmann, F., Selzer, J., Munro, J., Jeckel, D., and Aktories, K. (1998) J. Biol. Chem. 273, 19566-19572) and a conserved region defined by a tryptophan residue equivalent to Trp-102 of C. sordellii lethal toxin, From our findings, we propose a novel family of glycosyltransferases which includes both prokaryotic and eukaryotic proteins.