Colombo Maria-Luigi, Hanique Sophie, Baurin Stéphane L, Bauvois Cédric, De Vriendt Kris, Van Beeumen Jozef J, Frère Jean-Marie, Joris Bernard
Centre d'Ingénierie des Protéines, Institut de Chimie B6a, Université de Liège, Sart Tilman, B-4000 Liège 1, Belgium.
Antimicrob Agents Chemother. 2004 Feb;48(2):484-90. doi: 10.1128/AAC.48.2.484-490.2004.
The ybxI gene of Bacillus subtilis 168 encodes a preprotein of 267 amino acid residues, including a putative signal peptide of 23 residues. The YbxI primary structure exhibits high similarity scores with two members of the superfamily of the serine penicillin-recognizing enzymes: the class D beta-lactamases and the hydrophilic carboxy-terminal domains of the BlaR and MecR penicillin receptors. To determine the function and the activity of this putative penicillin-recognizing enzyme, we have subcloned the ybxI gene in the pET-26b expression vector. Transformation of Escherichia coli BL21(DE3) by the recombinant plasmid pCIP51 resulted in the export of the mature YbxI in the periplasm as a water-soluble protein. The recombinant protein was purified to 95% homogeneity. YbxI interacts with several beta-lactam antibiotics and can hydrolyze some of them. YbxI is not inactivated by clavulanic acid. The YbxI function and its enzymatic activity in B. subtilis remain unknown. The acyl-enzyme obtained after incubation of YbxI with a fluorescent derivative of ampicillin can be detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, confirming that YbxI can be acylated by beta-lactam antibiotics. YbxI does not hydrolyze some of the standard substrates of D-alanyl-D-alanine peptidases, the targets of penicillin. YbxI belongs to the penicillin-recognizing enzyme family but has an activity intermediate between those of a penicillin-binding protein and a beta-lactamase.
枯草芽孢杆菌168的ybxI基因编码一个由267个氨基酸残基组成的前体蛋白，其中包括一个由23个残基组成的假定信号肽。YbxI的一级结构与丝氨酸青霉素识别酶超家族的两个成员具有高度相似性得分：D类β-内酰胺酶以及BlaR和MecR青霉素受体的亲水性羧基末端结构域。为了确定这种假定的青霉素识别酶的功能和活性，我们将ybxI基因亚克隆到pET-26b表达载体中。用重组质粒pCIP51转化大肠杆菌BL21(DE3)，导致成熟的YbxI作为水溶性蛋白输出到周质中。重组蛋白被纯化至95%的纯度。YbxI与几种β-内酰胺抗生素相互作用，并能水解其中一些抗生素。YbxI不会被克拉维酸灭活。YbxI在枯草芽孢杆菌中的功能及其酶活性仍然未知。用氨苄青霉素的荧光衍生物孵育YbxI后得到的酰基酶可以通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳检测到，这证实了YbxI可以被β-内酰胺抗生素酰化。YbxI不会水解青霉素作用靶点D-丙氨酰-D-丙氨酸肽酶的一些标准底物。YbxI属于青霉素识别酶家族，但具有介于青霉素结合蛋白和β-内酰胺酶之间的活性。
