Wilde Christian, Vogelsgesang Martin, Aktories Klaus
Institut für Experimentelle und Klinische Pharmakologie und Toxikologie der Universität Freiburg, Otto-Krayer-Haus, Albertstrasse 25, D-79104 Freiburg, Germany.
Biochemistry. 2003 Aug 19;42(32):9694-702. doi: 10.1021/bi034583b.
C3-like ADP-ribosyltransferases represent an expanding family of related exoenzymes, which are produced by Clostridia and various Staphylococcus aureus strains. Here we report on the cloning and biochemical characterization of an ADP-ribosyltransferase from Bacillus cereus strain 2339. The transferase encompasses 219 amino acids; it has a predicted mass of 25.2 kDa and a theoretical isoelectric point of 9.3. To indicate the relationship to the family of C3-like ADP-ribosyltransferases, we termed the enzyme C3cer. The amino acid sequence of C3cer is 30 to 40% identical to other C3-like exoenzymes. By site-directed mutagenesis, Arg(59), Arg(97), Tyr(151), Arg(155), Thr(178), Tyr(180), Gln(183), and Glu(185) of recombinant C3cer were identified as pivotal residues of enzyme activity and/or protein substrate recognition. Precipitation experiments with immobilized RhoA revealed that C3cerTyr(180), which is located in the so-called "ADP-ribosylating toxin turn-turn" (ARTT) motif, plays a major role in the recognition of RhoA. Like other C3-like exoenzymes, C3cer ADP-ribosylates preferentially RhoA and RhoB and to a much lesser extent RhoC. Because the cellular accessibility of recombinant C3cer is low, a fusion toxin (C2IN-C3cer), consisting of the N-terminal 225 amino acid residues of the enzyme component of C2 toxin from Clostridium botulinum and C3cer was used to study the cytotoxic effects of the transferase. This fusion toxin caused rounding up of Vero cells comparable to the effects of Rho-inactivating toxins.
类C3 ADP核糖基转移酶是一个不断扩大的相关外切酶家族，由梭菌属和各种金黄色葡萄球菌菌株产生。在此，我们报告了蜡样芽孢杆菌菌株2339中一种ADP核糖基转移酶的克隆及生化特性。该转移酶包含219个氨基酸；预测分子量为25.2 kDa，理论等电点为9.3。为表明其与类C3 ADP核糖基转移酶家族的关系，我们将该酶命名为C3cer。C3cer的氨基酸序列与其他类C3外切酶的序列一致性为30%至40%。通过定点诱变，重组C3cer的精氨酸（59）、精氨酸（97）、酪氨酸（151）、精氨酸（155）、苏氨酸（178）、酪氨酸（180）、谷氨酰胺（183）和谷氨酸（185）被确定为酶活性和/或蛋白质底物识别的关键残基。用固定化RhoA进行的沉淀实验表明，位于所谓“ADP核糖基化毒素转折-转折”（ARTT）基序中的C3cer酪氨酸（180）在RhoA的识别中起主要作用。与其他类C3外切酶一样，C3cer优先对RhoA和RhoB进行ADP核糖基化，而对RhoC的作用程度要小得多。由于重组C3cer的细胞可及性较低，一种融合毒素（C2IN-C3cer）被用于研究该转移酶的细胞毒性作用，该融合毒素由肉毒梭菌C2毒素酶成分的N端225个氨基酸残基和C3cer组成。这种融合毒素导致Vero细胞变圆，其效果与Rho失活毒素相当。
